Modified fibroblast growth factor 21 (fgf-21) for use in methods for treating nonalcoholic steatohepatitis (nash)

ABSTRACT

Provided herein are methods for treating a patient having NASH who has been determined to have a particular threshold level of serum Pro-C3 (e.g., greater than 10 ng/ML) by administering to the patient a modified Fibroblast growth factor 21 (FGF-21) in an amount and with a frequency sufficient to treat NASH. Also provided are methods for monitoring responsiveness of a patient having NASH to treatment with a modified FGF-21, the method comprising: determining the serum Pro-C3 level in a blood sample from the patient obtained during or after treatment, wherein: a decreased serum Pro-C3 level in the blood sample from the patient obtained during or after treatment, as compared to the serum Pro-C3 level in a blood sample from the patient obtained prior to treatment with the modified FGF-21, indicates that the patient is responsive to treatment with the modified FGF-21.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/644,839, filed Mar. 5, 2020, pending, which application is a NationalStage Entry of International Application No. PCT/US2018/049729, filedSep. 6, 2018, which claims the benefit of priority of U.S. ProvisionalApplication Nos. 62/556,179, (filed on Sep. 8, 2017), 62/571,960 (filedon Oct. 13, 2017) and 62/640,211 (filed on Mar. 8, 2018). The contentsof the aforementioned applications are hereby incorporated by referencein their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Apr. 26, 2023, isnamed MXI-609USCN_SL.xml and is 5 KB in size.

BACKGROUND

Nonalcoholic fatty liver disease (NAFLD) has become one of the majordiseases plaguing the nation and world. In the United States, NAFLD isthe most common cause of liver disease, representing over 75% of thechronic liver disease (see, e.g., Younossi Z M, et al., Clin.Gastroenterol. Hepatol., 2011; 9:524-530). It also is one of the mostcommon indications for liver transplantation, contributing a majorburden to both the morbidity and mortality of the nation.

The spectrum of NAFLD is a continuum ranging from simple steatosis toNonalcoholic Steatohepatitis (NASH) and finally cirrhosis. The definingcharacteristic of the disease is the presence of greater than normallipid deposition within the liver with the absence of excessive alcoholconsumption defined as >20 g/d for men and 10 g/d for women. Steatosisis the presence of lipid within the cytoplasm of hepatocytes, thecriteria for which is defined in the literature as being either hepaticlipid levels above the 95th percentile for healthy individuals(about >55 mg/g liver) (see Cohen J C, et al., Science. 2011;332:1519-1523), greater than 5% of the liver's weight (see, KareemHassan, et al., World J. Gastroenterol. 2014 Sep. 14; 20(34):12082-12101), or found in greater than 5% of hepatocytes histologically(see Neuschwander-Tetri B A, Am. J. Med. Sci. 2005; 330:326-335).

The progression of steatosis to NASH is a frequently encounteredclinical scenario, associated with worse outcomes for patients. Akey-defining feature of the NASH is the presence of inflammation andsubsequent fibrosis. Specifically, NASH is defined as steatosis in thepresence of hepatocyte damage, inflammation and/or subsequent scarringand replacement of the tissue with type I collagen. Approximately10%-29% of patients with NASH will develop cirrhosis within a 10 yearperiod (Argo C K, et al., Clin. Liver Dis. 2009; 13:511-531). NASH canalso lead to liver cancer. Currently, no pharmacological therapies areapproved for the treatment of NASH (see Susanne Schuster and Ariel E.Feldstein, Nature Reviews Gastroenterology & Hepatology 14, 329-330 (5Apr. 2017).

Accordingly, the following disclosure provides a novel method oftreating NASH using a modified FGF-21, criteria that allow foroptimization of the treatment, and methods for monitoring theprogression and abatement of NASH.

SUMMARY

Provided herein are methods for treating a patient having NASH byadministering to the patient a modified Fibroblast growth factor 21(FGF-21) if the patient has a particular serum threshold level (e.g.,greater than 10 ng/ML, 15 ng/ML, or 20 ng/ML) of the biomarker, Pro-C3.

In one embodiment, a method for treating a patient having NASH isprovided, the method comprising: (a) determining the serum Pro-C3 levelin a blood sample from the patient and (b) administering to the patienta modified FGF-21 in an amount and with a frequency sufficient to treatNASH, if the serum Pro-C3 level prior to administration to the patientof the modified FGF-21 is greater than about 10 ng/ML.

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH.

In another embodiment, a method for treating a patient with NASH withmodified FGF-21 is provided, wherein the patient has a serum Pro-C3level greater than 10 ng/ML prior to treatment, wherein the methodcomprises administering to the patient a modified FGF-21 in an amountand with a frequency sufficient to treat NASH.

In another embodiment, a method for treating a patient having NASH isprovided, wherein the method comprises: (1) obtaining or having obtaineda blood sample from the patient, (2) determining or having determined aserum Pro-C3 level in the blood sample that is greater than 10 ng/ML,and (3) administering to the patient a modified FGF-21 in an amount andwith a frequency sufficient to treat NASH after a serum Pro-C3 level hasbeen determined.

Also provided are methods for monitoring responsiveness of a patienthaving NASH to treatment with a modified FGF-21, the method comprising:determining the serum Pro-C3 level in a blood sample from the patientobtained during or after treatment, wherein: a decreased serum Pro-C3level in the blood sample from the patient obtained during or aftertreatment, as compared to the serum Pro-C3 level in a blood sample fromthe patient obtained prior to treatment with the modified FGF-21,indicates that the patient is responsive to treatment with the modifiedFGF-21.

In one embodiment, the patient having NASH has been determined to haveor is determined to have a particular threshold level of Pro-C3 thatwarrants treatment with a modified FGF-21. In one embodiment, thepatient has a serum Pro-C3 level that is greater than 10 ng/ML. In oneembodiment, the patient has a serum Pro-C3 level that is greater than 11ng/ML. In one embodiment, the patient has a serum Pro-C3 level that isgreater than 12 ng/ML. In one embodiment, the patient has a serum Pro-C3level that is greater than 13 ng/ML. In one embodiment, the patient hasa serum Pro-C3 level that is greater than 14 ng/ML. In one embodiment,the patient has a serum Pro-C3 level that is greater than 15 ng/ML. Inone embodiment, the patient has a serum Pro-C3 level that is greaterthan 16 ng/ML. In one embodiment, the patient has a serum Pro-C3 levelthat is greater than 17 ng/ML. In one embodiment, the patient has aserum Pro-C3 level that is greater than 18 ng/ML. In one embodiment, thepatient has a serum Pro-C3 level that is greater than 19 ng/ML. In oneembodiment, the patient has a serum Pro-C3 level that is greater than 20ng/ML. In one embodiment, the patient has a serum Pro-C3 level that isgreater than 21 ng/ML. In one embodiment, the patient has a serum Pro-C3level that is greater than 22 ng/ML. In one embodiment, the patient hasa serum Pro-C3 level that is greater than 23 ng/ML. In one embodiment,the patient has a serum Pro-C3 level that is greater than 24 ng/ML. Inone embodiment, the patient has a serum Pro-C3 level that is greaterthan 25 ng/ML.

In another embodiment, the patient has a serum Pro-C3 level between 10ng/ML and 25 ng/ML. In another embodiment, the patient has a serumPro-C3 level between 10 ng/ML and 20 ng/ML. In another embodiment, thepatient has a serum Pro-C3 level between 10 ng/ML and 15 ng/ML. Inanother embodiment, the patient has a serum Pro-C3 level between 12ng/ML and 20 ng/ML. In another embodiment, the patient has a serumPro-C3 level between 12 ng/ML and 15 ng/ML. In another embodiment, thepatient has a serum Pro-C3 level between 15 ng/ML and 25 ng/ML. Inanother embodiment, the patient has a serum Pro-C3 level between 15ng/ML and 20 ng/ML.

Expression levels Pro-C3 can be measured by quantitation of proteinand/or RNA levels in a biological sample from the patient (e.g., bloodor a blood fraction) using any suitable technique. In one embodiment,expression levels are measured by quantitation of protein and/or RNAlevels, using at least one of an immunoassay, immunochemistry assay,immunohistochemistry assay, nucleoprobe assay, in situ hybridization,fluorescent RNA probes, RT-PCR, microarray transcription assay, and/orRNA transcription assay. In another embodiment, expression levels aremeasured using an immunoassay (e.g., an enzyme-linked immunosorbentassay (ELISA) or a radioimmunoassay (RIA)), e.g. the ELISA described inNielsen et al., Am J Transl Res 2013; 5(3):303-315. Pro-C3 level orlevels may be measured by an FDA-approved test.

Any suitable modified FGF-21 can be used in the method described herein.In one embodiment, the modified FGF-21 comprises the polypeptide of SEQID NO:1, except that an amino acid in the polypeptide is substituted bya non-naturally encoded amino acid, wherein: (a) said non-naturallyencoded amino acid is at a position corresponding to residue 108 of SEQID NO:1; (b) said non-naturally encoded amino acid comprises para-acetylphenylalanine linked to a polymer comprising a poly(ethylene glycol). Inanother embodiment, the poly(ethylene glycol) has an average molecularweight of about 30 kDa. In another embodiment, the modified FGF-21comprises the polypeptide of SEQ ID NO:1 except that the amino acid atposition 108 of SEQ ID NO:1 is substituted by a non-naturally encodedamino acid, wherein: (a) said non-naturally encoded amino acid comprisespara-acetyl phenylalanine, and (b) said non-naturally encoded amino acidis linked to a polymer comprising a poly(ethylene glycol) having anaverage molecular weight of about 30 kDa. In another embodiment, thenon-naturally encoded amino acid is linked to said polymer through anoxime linkage. In another embodiment, the modified FGF-21 comprises SEQID NO:2. In some embodiment, the para-acetyl phenylalanine in SEQ IDNO:2 is linked to a polymer comprising a poly(ethylene glycol). In someembodiment, the poly(ethylene glycol) has an average molecular weight ofabout 30 kDa.

In another embodiment a method for treating a patient having NASH isprovided, comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFGF-21 in an amount and with a frequency sufficient to treat NASH, ifthe serum Pro-C3 level prior to administration to the patient of amodified FGF-21 is greater than about 10 ng/ML (e.g. greater than about11 ng/ML, about 12 ng/ML, about 13 ng/ML, about 14 ng/ML, about 15ng/ML, about 16 ng/ML, about 17 ng/ML, about 18 ng/ML, about 19 ng/ML,about 20 ng/ML, about 21 ng/ML, about 22 ng/ML, about 23 ng/ML, about 24ng/ML, or about 25 ng/ML), wherein the modified FGF-21 comprises thepolypeptide of SEQ ID NO:1, except that an amino acid in the polypeptideis substituted by a non-naturally encoded amino acid, wherein: (a) saidnon-naturally encoded amino acid is at a position corresponding toresidue 108 of SEQ ID NO:1; and (b) said non-naturally encoded aminoacid comprises para-acetyl phenylalanine linked to a polymer comprisinga poly(ethylene glycol).

In another embodiment a method for treating a patient having NASH isprovided, comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFGF-21 in an amount and with a frequency sufficient to treat NASH, ifthe serum Pro-C3 level prior to administration to the patient of amodified FGF-21 is greater than about 10 ng/ML (e.g., greater than about11 ng/ML, about 12 ng/ML, about 13 ng/ML, about 14 ng/ML, about 15ng/ML, about 16 ng/ML, about 17 ng/ML, about 18 ng/ML, about 19 ng/ML,about 20 ng/ML, about 21 ng/ML, about 22 ng/ML, about 23 ng/ML, about 24ng/ML, or about 25 ng/ML), wherein the modified FGF-21 comprises thepolypeptide of SEQ ID NO:1 except that the amino acid at position 108 ofSEQ ID NO:1 is substituted by a non-naturally encoded amino acid,wherein: (a) said non-naturally encoded amino acid comprises para-acetylphenylalanine, and (b) said non-naturally encoded amino acid is linkedto a polymer comprising a poly(ethylene glycol) having an averagemolecular weight of about 30 kDa.

In another embodiment, a method for treating a patient having NASH isprovided, comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFGF-21 in an amount and with a frequency sufficient to treat NASH, ifthe serum Pro-C3 level prior to administration to the patient of amodified FGF-21 is greater than about 10 ng/ML (e.g. greater than about11 ng/ML, about 12 ng/ML, about 13 ng/ML, about 14 ng/ML, about 15ng/ML, about 16 ng/ML, about 17 ng/ML, about 18 ng/ML, about 19 ng/ML,about 20 ng/ML, about 21 ng/ML, about 22 ng/ML, about 23 ng/ML, about 24ng/ML, or about 25 ng/ML), wherein the modified FGF-21 consists of orcomprises SEQ ID NO:2. In some embodiment, the para-acetyl phenylalaninein SEQ ID NO:2 is linked to a polymer comprising a poly(ethyleneglycol). In some embodiments, the poly(ethylene glycol) has an averagemolecular weight of about 30 kDa.

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH, wherein the modified FGF-21 comprises the polypeptide of SEQ IDNO:1, except that an amino acid in the polypeptide is substituted by anon-naturally encoded amino acid, wherein: (a) said non-naturallyencoded amino acid is at a position corresponding to residue 108 of SEQID NO:1; and (b) said non-naturally encoded amino acid comprisespara-acetyl phenylalanine linked to a polymer comprising a poly(ethyleneglycol).

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH, wherein the modified FGF-21 comprises the polypeptide of SEQ IDNO:1 except that the amino acid at position 108 of SEQ ID NO:1 issubstituted by a non-naturally encoded amino acid, wherein: (a) saidnon-naturally encoded amino acid comprises para-acetyl phenylalanine,and (b) said non-naturally encoded amino acid is linked to a polymercomprising a poly(ethylene glycol) having an average molecular weight ofabout 30 kDa.

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH, wherein the modified FGF-21 consists of or comprises SEQ ID NO:2.In some embodiments, the para-acetyl phenylalanine in SEQ ID NO:2 islinked to a polymer comprising a poly(ethylene glycol). In someembodiments, the poly(ethylene glycol) has an average molecular weightof about 30 kDa.

In some embodiments described herein, the patient having NASH has beendetermined to have a serum Pro-C3 level greater than about 11 ng/ML,about 12 ng/ML, about 13 ng/ML, about 14 ng/ML, about 15 ng/ML, about 16ng/ML, about 17 ng/ML, about 18 ng/ML, about 19 ng/ML, about 20 ng/ML,about 21 ng/ML, about 22 ng/ML, about 23 ng/ML, about 24 ng/ML, or about25 ng/ML.

In another embodiment, a method for monitoring responsiveness of apatient having NASH to treatment with a modified FGF-21 is provided, themethod comprising: determining the serum Pro-C3 level in a blood samplefrom the patient obtained during or after treatment, wherein: adecreased serum Pro-C3 level in the blood sample from the patientobtained during or after treatment, as compared to the serum Pro-C3level in a blood sample from the patient obtained prior to treatmentwith the modified FGF-21, indicates that the patient is responsive totreatment with the modified FGF-21, wherein the modified FGF-21comprises the polypeptide of SEQ ID NO:1, except that an amino acid inthe polypeptide is substituted by a non-naturally encoded amino acid,wherein: (a) said non-naturally encoded amino acid is at a positioncorresponding to residue 108 of SEQ ID NO:1; and (b) said non-naturallyencoded amino acid comprises para-acetyl phenylalanine linked to apolymer comprising a poly(ethylene glycol)

In another embodiment, a method for monitoring responsiveness of apatient having NASH to treatment with a modified FGF-21 is provided, themethod comprising: determining the serum Pro-C3 level in a blood samplefrom the patient obtained during or after treatment, wherein: adecreased serum Pro-C3 level in the blood sample from the patientobtained during or after treatment, as compared to the serum Pro-C3level in a blood sample from the patient obtained prior to treatmentwith the modified FGF-21, indicates that the patient is responsive totreatment with the modified FGF-21, wherein the modified FGF-21comprises the polypeptide of SEQ ID NO:1 except that the amino acid atposition 108 of SEQ ID NO:1 is substituted by a non-naturally encodedamino acid, wherein: (a) said non-naturally encoded amino acid comprisespara-acetyl phenylalanine, and (b) said non-naturally encoded amino acidis linked to a polymer comprising a poly(ethylene glycol) having anaverage molecular weight of about 30 kDa.

In another embodiment, a method for monitoring responsiveness of apatient having NASH to treatment with a modified FGF-21 is provided, themethod comprising: determining the serum Pro-C3 level in a blood samplefrom the patient obtained during or after treatment, wherein: adecreased serum Pro-C3 level in the blood sample from the patientobtained during or after treatment, as compared to the serum Pro-C3level in a blood sample from the patient obtained prior to treatmentwith the modified FGF-21, indicates that the patient is responsive totreatment with the modified FGF-21, wherein the modified FGF-21 consistsof or comprises SEQ ID NO:2. In some embodiments, the para-acetylphenylalanine in SEQ ID NO:2 is linked to a polymer comprising apoly(ethylene glycol). In some embodiments, the poly(ethylene glycol)has an average molecular weight of about 30 kDa.

In one embodiment, the modified FGF-21 is administered at a fixed dose.In one embodiment, the modified FGF-21 is administered at a fixed dailyor weekly dose.

In one embodiment, a method for treating a patient having NASH isprovided, the method comprising administering to the patient a modifiedFGF-21 at 10 mg once daily to treat NASH, wherein the modified FGF-21comprises the polypeptide of SEQ ID NO:1 except that the amino acid atposition 108 of SEQ ID NO:1 is substituted by a non-naturally encodedamino acid, wherein: (a) said non-naturally encoded amino acid comprisespara-acetyl phenylalanine, and (b) said non-naturally encoded amino acidis linked to a polymer comprising a poly(ethylene glycol) having anaverage molecular weight of about 30 kDa.

In one embodiment, a method for treating a patient having NASH isprovided, the method comprising administering to the patient a modifiedFGF-21 at 20 mg once weekly to treat NASH, wherein the modified FGF-21comprises the polypeptide of SEQ ID NO:1 except that the amino acid atposition 108 of SEQ ID NO:1 is substituted by a non-naturally encodedamino acid, wherein: (a) said non-naturally encoded amino acid comprisespara-acetyl phenylalanine, and (b) said non-naturally encoded amino acidis linked to a polymer comprising a poly(ethylene glycol) having anaverage molecular weight of about 30 kDa.

In another embodiment, the modified FGF-21 is administered at a dose ofabout 10 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 11 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 12 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 13 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 14 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 15 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 16 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 17 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 18 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 19 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 20 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 21 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 22 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 23 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 24 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 25 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 26 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 27 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 28 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 29 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 30 mg once weekly.

In another embodiment, the modified FGF-21 is administered at a fixeddaily dose. In another embodiment, the modified FGF-21 is administeredat a dose of about 5 mg once daily. In another embodiment, the modifiedFGF-21 is administered at a dose of about 10 mg once daily. In anotherembodiment, the modified FGF-21 is administered at a dose of about 11 mgonce daily. In another embodiment, the modified FGF-21 is administeredat a dose of about 12 mg once daily. In another embodiment, the modifiedFGF-21 is administered at a dose of about 13 mg once daily. In anotherembodiment, the modified FGF-21 is administered at a dose of about 14 mgonce daily. In another embodiment, the modified FGF-21 is administeredat a dose of about 15 mg once daily. In another embodiment, the modifiedFGF-21 is administered at a dose of about 16 mg once daily. In anotherembodiment, the modified FGF-21 is administered at a dose of about 17 mgonce daily. In another embodiment, the modified FGF-21 is administeredat a dose of about 18 mg once daily. In another embodiment, the modifiedFGF-21 is administered at a dose of about 19 mg once daily. In anotherembodiment, the modified FGF-21 is administered at a dose of about 20 mgonce daily.

The modified FGF-21 can be administered according to the methodsdescribed herein alone or in combination with one or more additionaltherapeutic agents. In some embodiments, the one or more additionaltherapeutic agents may be selected from anti-fibrotic agents, N-cadherinantagonist, anti-N cadherin antibody, small molecule N-cadherinantagonist, antagonistic N-cadherin fragment, anti-inflammatory agents,hepatoprotective agents suppressing renin-angiotensin system (RAS)system, probiotics, and polyunsaturated fatty acids (PUFAs). In someembodiments, the anti-fibrotic agent may be selected from nintedanib,Pirfenidone, LPA1 antagonists, LPA1 receptor antagonists, GLP1 analog,tralokinumab (IL-13, AstraZeneca), vismodegib (hedgehog antagonist,Roche), PRM-151 (pentraxin-2, TGF beta-1, Promedior), SAR-156597(bispecific Mab IL-4&IL-13, Sanofi), simtuzumab (anti-lysyl oxidase-like2 (anti-LOXL2) antibody, Gilead), CKD-942, PTL-202 (PDEinh./pentoxifylline/NAC oral control. release, Pacific Ther.),omipalisib (oral PI3K/mTOR inhibitor, GSK), IW-001 (oral sol. bovinetype V collagen mod., ImmuneWorks), STX-100 (integrin alpha V/beta-6ant, Stromedix/Biogen), Actimmune (IFN gamma), PC-SOD (midismase;inhaled, LTT Bio-Pharma/CKD Pharm), lebrikizumab (anti-IL-13 SChumanized mAb, Roche). AQX-1125 (SHIP1 activator, Aquinox), CC-539 (JNKinhibitor, Celgene), FG-3019 (FibroGen), and SAR-100842 (Sanofi). Insome embodiments, the hepatoprotective agent may be ursodeoxycholic acid(UDCA) or obeticholic acid (OCA or INT-747, Intercept).

In one embodiment, the modified FGF-21 is administered first in time andthe one or more additional active agents are administered second intime. In some embodiments, the one or more additional active agents areadministered first in time and the modified FGF-21 is administeredsecond in time.

In one embodiment, the treatment methods described herein result in adecrease in Pro-C3 levels in a patient. In another embodiment, thetreatment methods described herein produce a shift toward normal levelsof Pro-C3 in a patient. In another embodiment, the treatment methodsdescribed herein result in a reduction in liver stiffness in a patient,as assessed by magnetic resonance elastography (MRE). For example, inone embodiment, treatment with a modified FGF-21 results in a reductionin liver stiffness in the patient compared to the patient's liverstiffness prior to treatment, wherein liver stiffness is assessed byMRE. In another embodiment, treatment with a modified FGF-21 results ina 15% or greater (e.g., 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 50%, 55%, 60%, 65%, 70%, 75% orgreater) reduction in liver stiffness in the patient compared to thepatient's liver stiffness prior to treatment, wherein liver stiffness isassessed by MRE.

In another embodiment, the treatment methods described herein result ina reduction in hepatic fat fraction in a patient. For example, in oneembodiment, treatment with a modified FGF-21 results in a reduction inhepatic fat fraction in the patient compared to the patient's hepaticfat fraction prior to treatment, wherein hepatic fat fraction is asassessed by magnetic resonance imaging-estimated proton density fatfraction (MRI-PDFF). In another embodiment, treatment with the modifiedFGF-21 results in a 30% or greater (e.g., 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75% orgreater) reduction in hepatic fat fraction in the patient compared tothe patient's hepatic fat fraction prior to treatment, wherein hepaticfat fraction is assessed by MRI-PDFF.

In another embodiment, the treatment methods described herein result ina reduction in live stiffness, a reduction in hepatic fat fraction,and/or a decrease in serum Pro-C3 levels in a patient. In anotherembodiment, treatment with a modified FGF-21 results in a reduction inliver stiffness in the patient compared to the patient's liver stiffnessprior to treatment, and a decrease in serum Pro-C3 levels in the patientcompared to the patient's serum Pro-C3 levels prior to treatment,wherein liver stiffness is assessed by MRE, and wherein the patient hasbeen determined to have a serum Pro-C3 level greater than 10, 15 or 20ng/ML prior to treatment with the modified FGF-21.

In another embodiment, the treatment methods described herein producesat least one therapeutic effect in a patient selected from the groupconsisting of a reduction or cessation in fatigue, malaise, weight loss,and/or right upper quadrant abdominal discomfort in the patient.

Also provided herein is a modified FGF-21 for use in the methodsdescribed herein. In one embodiment, a modified FGF-21 for use in amethod of treating NASH in a patient is provided, wherein the methodcomprises administering to the patient a modified FGF-21 in an amountand with a frequency sufficient to treat NASH, wherein the patient hasbeen determined to have a serum Pro-C3 level greater than 10, 15 or 20ng/ML. In another embodiment, a modified FGF-21 for use in a method ofmonitoring responsiveness of a patient having NASH to treatment with amodified FGF-21 is provided, wherein a decreased serum Pro-C3 level in ablood sample from the patient obtained during or after treatment, ascompared to a serum Pro-C3 level in a blood sample from the patientobtained prior to treatment with the modified FGF-21, indicates that thepatient is responsive to treatment with the modified FGF-21.

Finally, methods of identifying a patient having NASH that is suitablefor treatment with a modified FGF-21 are provided, wherein the methodscomprise determining a serum Pro-C3 level in a blood sample from thepatient using an in vitro assay, wherein the serum Pro-C3 level in theblood sample is greater than 10, 15 or 20 ng/ML. Other features andadvantages of the methods of treatment will be apparent from thefollowing description, the examples, and from the claims. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the patient disposition for the trial.

FIGS. 2A-2C depict the change in hepatic fat fraction measured byMRI-PDFF at week 16. FIG. 2A depicts the absolute change in hepaticfraction in patients from baseline to week 16. FIG. 2B shows theMRI-PDFF measurements of a patient who experienced a reduction inhepatic fat fraction following BMS-986036 treatment. FIG. 2C depicts thepercent of patients with ≥30%, ≥20%, or ≥10% relative reductions inhepatic fat fraction.

FIGS. 3A-3B depict the change in liver stiffness in patients at week 16.FIG. 3A shows the absolute change in liver stiffness (MRE) from baselineto Week 16 and FIG. 3B shows the percent of patients with ≥15% relativereduction in liver stiffness (MRE) at week 16.

FIG. 4 is an MRE image from a patient treated with 20 mg QW BMS-986036.

FIG. 5 shows the percent change in adiponectin from baseline to week 16.

FIGS. 6A-6B show baseline Pro-C3 distribution among BMS-986036-treatedpatients.

FIG. 6A shows Pro-C3 levels for individuals treated with BMS-986036.FIG. 6B shows the frequency of Pro-C3 levels.

FIGS. 7A-7B show the reduction in Serum Pro-C3 at Week 16. FIG. 7A showsthe percent change from baseline PRO-C3 levels at week 16. FIG. 7B showsthe percent of patients with ≥15% relative reduction in PRO-C3 at week16. Inferential statistical analyses were conducted post hoc using alongitudinal repeated measurements model analysis. ^(a)Sample size forserum Pro-C3 was smaller than MRI-PDFF due to some non-evaluable samplesat baseline.

FIGS. 8A-8B show the changes in markers of liver injury at the end oftreatment.

FIG. 8A shows the percent change in ALT from baseline to week 16. FIG.8B shows the percent change in AST from baseline to week 16. BMS-986036QD and QW treatment were associated with improvements from baseline inbiomarkers of liver injury (n indicates number of patients with ALT/ASTdata at end of treatment (EOT)).

FIGS. 9A-9C show the change in LDL cholesterol (FIG. 9A), HDLcholesterol (FIG. 9B), and triglycerides (FIG. 9C) from baseline to Week16.

FIGS. 10A-10C show the bone mineral density measured by DXA at weeks 16(D112) and 6 months post-treatment (D292) in the femur (FIG. 10A), hip(FIG. 10B), and spine (FIG. 10C).

FIGS. 11A-11B show the change in MRE (FIG. 11A) and Pro-C3 (FIG. 11B) atweek 16 in patients stratified by baseline Pro-C3 (>=20 ng/ML and <20ng/ML).

FIGS. 12A-12D show the changes in Pro-C3 (FIG. 12A), ALT (FIG. 12B), AST(FIG. 12C), and CK-18 (FIG. 12D) at week 16 in patients stratified byreduction in hepatic fat fraction.

FIGS. 13A-13C show the changes in Pro-C3 (FIG. 13A), HA (FIG. 13B), andHDL (FIG. 13C) at week 16 in patients stratified by reduction in liverstiffness.

DETAILED DESCRIPTION

Provided herein are methods for treating a patient having NASH byadministering to the patient a modified FGF-21 if the patient has beendetermined to have a particular serum threshold level of Pro-C3 (e.g.,greater than about 10 ng/ML, greater than 15 ng/ML, or greater thanabout 17 ng/ML, or greater than about 20 ng/ML) prior to administrationto the patient of a modified FGF-21, as well as methods for monitoringresponsiveness of a patient having NASH to treatment with a modifiedFGF-21 based on serum levels of Pro-C3.

I. Definitions

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art, andconventional methods of immunology, protein chemistry, biochemistry,recombinant DNA techniques, and pharmacology are employed.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. The use of “or”or “and” means “and/or” unless stated otherwise. Furthermore, use of theterm “including” as well as other forms, such as “include”, “includes”,and “included”, is not limiting.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration and the like, is encompassesvariations of up to ±10% from the specified value. Unless otherwiseindicated, all numbers expressing quantities of ingredients, propertiessuch as molecular weight, reaction conditions, etc., used herein are tobe understood as being modified by the term “about”.

As used herein, the term “normal,” when used to modify the term“individual” or “subject” refers to an individual or group ofindividuals who does/do not have a particular disease or condition(e.g., NASH) and is also not suspected of having or being at risk fordeveloping the disease or condition. The term “normal” is also usedherein to qualify a biological specimen or sample (e.g., a blood or afraction thereof) isolated from a normal or healthy individual orsubject (or group of such subjects), for example, a “normal controlsample” or “normal control biological fluid”.

“Polypeptide,” “peptide,” and “protein” are used interchangeably andmean any peptide-linked chain of amino acids, regardless of length orpost-translational modification. The proteins described herein cancontain or be wild-type proteins or can be variants that have not morethan 50 (e.g., not more than one, two, three, four, five, six, seven,eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative aminoacid substitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine.

As used herein, percent (%) amino acid sequence identity is defined asthe percentage of amino acids in a candidate sequence that are identicalto the amino acids in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST software. Appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared can be determinedby known methods.

The term “pharmaceutical formulation” or “pharmaceutical composition”refers to preparations which are in such form as to permit thebiological activity of the active ingredients to be unequivocallyeffective, and which contain no additional components which aresignificantly toxic to the subjects to which the formulation would beadministered.

As used herein, an “aqueous” pharmaceutical composition is a compositionsuitable for pharmaceutical use, wherein the aqueous carrier is water. Acomposition suitable for pharmaceutical use may be sterile, homogeneousand/or isotonic. Aqueous pharmaceutical compositions may be prepareddirectly in an aqueous form and/or may be reconstituted from alyophilisate.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Preferred methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the presently disclosed methods and compositions. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor treating NASH in a subject, will be apparent from the followingdescription, the examples, and from the claims.

1. Pro-C3

Pro-C3 levels above a particular threshold level (e.g., greater thanabout 10 ng/ML, greater than 15 ng/ML, or greater than 20 ng/ML) can beused as an indicator to evaluate whether a patient having NASH will beresponsive to treatment with a modified FGF-21 (such as BMS-986036)and/or to monitor response to treatment with a modified FGF-21.

Pro-C3 (also known as “true collagen type III formation”) is a marker oftrue type III collagen formation. Together with type I collagen, typeIII collagen constitutes the major structural proteins in the humanbody, in which type III collagen is crucial for type I collagenfibrillogenesis except in bones, which almost exclusively consist oftype I collagen (see Bao X, et al., J Genet Genomics. 2007; 34:223-228;Jensen L T, et al., Cardiovasc Res. 1997; 33:535-539; and Nielsen etal., Am J Transl Res. 2013; 5(3): 303-315). During fibrillar assemblythe N-terminal propeptide of type III collagen is cleaved off byspecific N-proteases prior to incorporation of the mature collagen inthe extracellular matrix (ECM), thus released in the ECM and intocirculation. The propeptide molecule consists of three identicalα-chains with a total molecular weight of 42 kDa. The GenBank (NationalCenter for Biotechnology Information (NCBI)) reference numbers for theprotein and gene sequences of Pro-C3 (Collagen α-1 (III) chain) areNP_000081 (UniProt P02461) and NM_000090.3, respectively, the sequencesof which are expressly incorporated herein by reference. Fragments ofthe protein, for example, the N-terminal propeptide, such as amino acidsequence 144-CPTGPQNYSP-153 (SEQ ID NO: 3), in the α1 chain Pro-C3, maybe used to generate antibodies for immunoassay (e.g., ELISA). Nielsen etal., Am J Transl Res. 2013; 5(3): 303-315.

Measuring or determining protein levels of a biomarker, such as Pro-C3,in a biological sample may be performed by any suitable method (see,e.g., Harlow and Lane (1988) “Antibodies: A Laboratory Manual”, ColdSpring Harbor Laboratory: Cold Spring Harbor, NY).

In general, protein levels are determined by contacting a biologicalsample obtained from a subject with binding agents for one or more ofthe biomarker proteins; detecting, in the biological sample theexpression level (e.g., levels) of one or more of the biomarker proteinsthat bind to the binding agents; and comparing the levels of one or moreof the biomarker proteins in the sample with the levels of thecorresponding protein biomarkers in a control sample (e.g., a normalsample).

Suitable binding agents also include an antibody specific for abiomarker protein described herein (e.g., Pro-C3). Suitable antibodiesfor use in the methods of the present invention include monoclonal andpolyclonal antibodies and antigen-binding fragments (e.g., Fab fragmentsor scFvs) of antibodies. Antibodies, including monoclonal and polyclonalantibodies, fragments and chimeras, may be prepared using methods knownin the art (see, for example, Kohler and Milstein (1975) Nature256:495-497; Kozbor et al. (1985) J Immunol Methods 81:31-42; Cote etal. (1983) Proc Natl Acad Sci USA 80:2026-203; and Zhang et al. (2002) JBiol Chem 277:39379-39387). Exemplary antibodies include antibodies orfragments thereof that bind to the N-terminal propeptide, such as aminoacid sequence 144-CPTGPQNYSP-153 (SEQ ID NO: 3), in the α1 chain Pro-C3(e.g., antibody NB61N-62, as described in Nielsen et al., Am J TranslRes. 2013; 5(3): 303-315). Antibodies to be used in the methods of theinvention can be purified by methods well known in the art. Antibodiesmay also be obtained from commercial sources.

In certain embodiments, the binding agent is directly or indirectlylabeled with a detectable moiety. The role of a detectable agent is tofacilitate the detection step of the diagnostic method by allowingvisualization of the complex formed by binding of the binding agent tothe protein marker (or fragment thereof). The detectable agent can beselected such that it generates a signal that can be measured and whoseintensity is related (preferably proportional) to the amount of proteinmarker present in the sample being analyzed. Methods for labelingbiological molecules such as polypeptides and antibodies are well-knownin the art. Any of a wide variety of detectable agents can be used inthe practice of the present invention. Suitable detectable agentsinclude, but are not limited to: various ligands, radionuclides,fluorescent dyes, chemiluminescent agents, microparticles (such as, forexample, quantum dots, nanocrystals, phosphors and the like), enzymes(such as, e.g., those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, digoxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

In certain embodiments, the binding agents (e.g., antibodies) may beimmobilized on a carrier or support (e.g., a bead, a magnetic particle,a latex particle, a microtiter plate well, a cuvette, or other reactionvessel). Examples of suitable carrier or support materials includeagarose, cellulose, nitrocellulose, dextran, Sephadex®, Sepharose®,liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene,gabbros, filter paper, magnetite, ion-exchange resin, plastic film,plastic tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer,amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, andthe like. Binding agents may be indirectly immobilized using secondbinding agents specific for the first binding agents (e.g., mouseantibodies specific for the protein markers may be immobilized usingsheep anti-mouse IgG Fc fragment specific antibody coated on the carrieror support).

Protein expression levels in a biological sample may be determined usingimmunoassays. Examples of such assays are time resolved fluorescenceimmunoassays (TR-FIA), radioimmunoassays, enzyme immunoassays (e.g.,ELISA), immunofluorescence immunoprecipitation, latex agglutination,hemagglutination, Western blot, and histochemical tests, which areconventional methods well-known in the art. Methods of detection andquantification of the signal generated by the complex formed by bindingof the binding agent with the protein marker will depend on the natureof the assay and of the detectable moiety (e.g., fluorescent moiety). Anexemplary Pro-C3 ELISA is described in Nielson et al., Am J Transl Res2013; 5(3):303-315, which is incorporated herein by reference.

In one example, the presence or amount of protein expression of a gene(e.g., a biomarker, such as Pro-C3) can be determined using a Westernblotting technique. For example, a lysate can be prepared from abiological sample, or the biological sample (e.g., blood or a fractionthereof) itself, can be contacted with Laemmli buffer and subjected tosodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).SDS-PAGE-resolved proteins, separated by size, can then be transferredto a filter membrane (e.g., nitrocellulose) and subjected toimmunoblotting techniques using a detectably-labeled antibody specificto the protein of interest. The presence or amount of bounddetectably-labeled antibody indicates the presence or amount of proteinin the biological sample.

In another example, an immunoassay can be used for detecting and/ormeasuring the protein expression of a biomarker protein, such as Pro-C3.As above, for the purposes of detection, an immunoassay can be performedwith an antibody that bears a detection moiety (e.g., a fluorescentagent or enzyme). Proteins from a biological sample can be conjugateddirectly to a solid-phase matrix (e.g., a multi-well assay plate,nitrocellulose, agarose, Sepharose®, encoded particles, or magneticbeads) or it can be conjugated to a first member of a specific bindingpair (e.g., biotin or streptavidin) that attaches to a solid-phasematrix upon binding to a second member of the specific binding pair(e.g., streptavidin or biotin). Such attachment to a solid-phase matrixallows the proteins to be purified away from other interfering orirrelevant components of the biological sample prior to contact with thedetection antibody and also allows for subsequent washing of unboundantibody. Here, as above, the presence or amount of bounddetectably-labeled antibody indicates the presence or amount of proteinin the biological sample.

Alternatively, the protein expression levels may be determined usingmass spectrometry based methods or image-based methods known in the artfor the detection of proteins. Other suitable methods include 2D-gelelectrophoresis, proteomics-based methods such as the identification ofindividual proteins recovered from the gel (e.g., by mass spectrometryor N-terminal sequencing) and/or bioinformatics.

Methods for detecting or measuring protein expression can, optionally,be performed in formats that allow for rapid preparation, processing,and analysis of multiple samples. This can be, for example, inmulti-well assay plates (e.g., 96 wells or 386 wells) or arrays (e.g.,protein chips). Stock solutions for various reagents can be providedmanually or robotically, and subsequent sample preparation, pipetting,diluting, mixing, distribution, washing, incubating (e.g.,hybridization), sample readout, data collection (optical data) and/oranalysis (computer aided image analysis) can be done robotically usingcommercially available analysis software, robotics, and detectioninstrumentation capable of detecting the signal generated from theassay. Examples of such detectors include, but are not limited to,spectrophotometers, luminometers, fluorimeters, and devices that measureradioisotope decay. Exemplary high-throughput cell-based assays (e.g.,detecting the presence or level of a target protein in a cell) canutilize ArrayScan® VTI HCS Reader or KineticScan® HCS Reader technology(Cellomics Inc., Pittsburgh, PA).

Expression of the biomarker can also be detected at the nucleic acidlevel (e.g., based on RNA levels). In one embodiment, RNA is detectedusing an RNA-ISH assay. Another method for determining the level of RNAin a sample involves the process of nucleic acid amplification fromhomogenized tissue, e.g., by RT-PCR (reverse transcribing the RNA andthen, amplifying the resulting cDNA employing PCR or any other nucleicacid amplification method, followed by the detection of the amplifiedmolecules. In another embodiment, RNA expression is assessed byquantitative fluorogenic RT-PCR (qPCR).

In one embodiment, the methods described herein involve comparing themeasured expression level or activity of a biomarker protein, such asPro-C3 (as measured in a biological sample obtained from a patient) to acontrol sample. In some embodiments, control sample is obtained from thepatient prior to administering to the patient the modified FGF-21 (e.g.,BMS-986036). In some embodiments, the control sample can be (or can bebased on), e.g., a collection of samples obtained from one or more(e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25,30, 35, or 40 or more) healthy individuals that have not beenadministered modified FGF-21. In some embodiments, the control samplecan be (or can be based on), e.g., a pooled sample obtained from two ormore (e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20,25, 30, 35, or 40 or more) individuals. In some embodiments of any ofthe methods described herein, the pooled samples can be from healthyindividuals, or at least, individuals who do not have or are notsuspected of having NASH. In another embodiment, determining whether theexpression level or activity of a biomarker, such as Pro-C3, or hepaticfat fraction, has decreased following treatment with a modified FGF-21can involve comparing the expression level or activity of the biomarkerin a biological sample obtained from a patient prior to treatment to theexpression level of the biomarker in a sample of the same biologicaltype obtained from the patient after treatment with the modified FGF-21(e.g., one day, two days, three days, four days, five days, six days, 1week, 2 weeks, 3 weeks, a month, 6 weeks, two months, three months, fourmonths, 5 months, or 6 months after treatment).

In some embodiments, determining whether a modified FGF-21 has produceda desired effect (e.g., a reduction in serum Pro-C3 levels (e.g., byabout 5, 10, 15, 20, 25, 30, 40, or 50%) and/or liver stiffness) in ahuman can be performed by querying whether the post-treatment expressionlevel of the biomarker (e.g., Pro-C3) falls within a predetermined rangeindicative of responsiveness to a modified FGF-21 by a human. In someembodiments, determining whether a modified FGF-21 has produced adesired effect in a human can include querying if the post-treatmentexpression level or activity of the biomarker (e.g., Pro-C3) falls aboveor below a predetermined cut-off value. A cut-off value is typically theexpression level or activity of a given biomarker in a given biologicalsample above or below which is considered indicative of a certainphenotype, e.g., responsiveness to therapy with a modified FGF-21.

In some embodiments of any of the methods described herein, the samepractitioner may administer the modified FGF-21 to the patient prior todetermining whether a change in the expression level or activity of thebiomarker (e.g., Pro-C3) has occurred, whereas in some embodiments, thepractitioner who administers the modified FGF-21 to the patient isdifferent from the practitioner who determines whether a response hasoccurred in the patient. In some embodiments, the practitioner mayobtain a biological sample from the patient prior to administration ofthe modified FGF-21. In some embodiments, the practitioner may obtain abiological sample from the patient following the administration of themodified FGF-21 to the patient. In some embodiments, the post-treatmentsample can be obtained from the patient less than 48 (e.g., less than47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, nine, eight, seven, six, five, four, three, two, or even lessthan one) hours following administration of the modified FGF-21 to thepatient. In some embodiments, the post-treatment sample can be obtainedfrom the patient less than 20 (e.g., less than 19, 18, 17, 16, 15, 14,13, 12, 11, 10, nine, eight, seven, six, five, four, three, two, or one)day(s) after administering to the patient the modified FGF-21. In someembodiments, the biological sample is obtained from the patient no morethan 20 (e.g., no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,nine, eight, seven, six, five, four, three, two, or one) day(s) afterthe modified FGF-21 is administered to the patient.

In some embodiments, the serum level of Pro-C3 is decreased by at least5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70)% following administration of the modified FGF-21. In some embodiments,the serum level of Pro-C3 is decreased by at least 15% followingadministration of the modified FGF-21. In some embodiments, the serumlevel of Pro-C3 is decreased by at least 20% following administration ofthe modified FGF-21.

In some embodiments, the serum level of Pro-C3 is decreased to within 90(e.g., 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30) % of thenormal serum level of Pro-C3 following administration of the modifiedFGF-21.

In some embodiments, the serum level of Pro-C3 is decreased to within 90(e.g., 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30) % of thepre-treatment (prior to treatment) Baseline serum level of Pro-C3following administration of the modified FGF-21.

2. Modified FGF-21 Polypeptides

As described in U.S. Pat. No. 9,434,778 (the contents of which areexpressly incorporated reference herein in its entirety), fibroblastgrowth factors are polypeptides widely expressed in developing and adulttissues (Baird et al., Cancer Cells, 3:239-243, 1991) that play crucialroles in multiple physiological functions (McKeehan et al., Prog.Nucleic Acid Res. Mol. Biol. 59:135-176, 1998; Burgess, W. H. et al.,Annu. Rev. Biochem. 58:575-606 (1989). According to the literature, theFGF family consists of at least twenty-two members (Reuss et al., CellTissue Res. 313:139-157 (2003)).

Fibroblast growth factor 21 (FGF-21) has been described in theliterature (Nishimura et al., Biochimica et Biophysica Acta,1492:203-206 (2000); WO 01/36640; and WO 01/18172, and U.S. PatentPublication No. 20040259780, each of which is expressly incorporated byreference herein in its entirety). Unlike other FGFs, FGF-21 has beenreported not to have proliferative and tumorigenic effects (Ornitz andItoh, Genome Biology 2001, 2(3):reviews 3005.1-3005.12).

Certain FGF-21 polypeptides and uses thereof are described in U.S.Patent Publication No. 20010012628, U.S. Pat. No. 6,716,626, U.S. PatentPublication No. 2004/0259780, WO 03/011213, Kharitonenkov et al. J ClinInvest. 2005 June; 115(6):1627-35, WO 03/059270, U.S. Patent PublicationNo. 2005/0176631, WO 2005/091944, WO 2007/0293430, U.S. PatentPublication No. 2007/0293430, WO/2008/121563, U.S. Pat. No. 4,904,584,WO 99/67291, WO 99/03887, WO 00/26354, and U.S. Pat. No. 5,218,092 eachof which is incorporated by reference herein in its entirety.

As used herein, “modified FGF-21 polypeptide,” “modified fibroblastgrowth factor 21” or “modified FGF-21” and unhyphenated forms thereofare used interchangeably and shall include those polypeptides andproteins that differ from wild-type FGF-21 (e.g., wild-type human FGF-21of SEQ ID NO:1) and typically have at least one biological activity of afibroblast growth factor 21, as well as FGF-21 analogs, FGF-21 isoforms,FGF-21 mimetics, FGF-21 fragments, hybrid FGF-21 proteins, fusionproteins, oligomers and multimers, homologues, glycosylation patternvariants, variants, splice variants, and muteins, thereof, regardless ofthe biological activity of same. The term “modified FGF-21 polypeptide”and “modified FGF-21” encompass FGF-21 polypeptides comprising one ormore amino acid substitutions, additions or deletions. For example,modified FGF-21 polypeptides of the present disclosure may comprise oneor more non-natural amino acid modification, optionally in conjunctionwith modifications with one or more natural amino acids. Exemplarysubstitutions, insertions or deletions in a wide variety of amino acidpositions in FGF-21 polypeptides (including those described herein andothers), including but not limited to substitutions that modulatepharmaceutical stability, that modulate one or more of the biologicalactivities of the FGF-21 polypeptide, such as but not limited to,increase agonist activity, increase solubility of the polypeptide,decrease protease susceptibility, decrease deamidation, convert thepolypeptide into an antagonist, reduce immunogenicity or toxicity, orfacilitate purification or manufacturability, etc., are encompassed bythe term “modified FGF-21 polypeptide.”

In some embodiments, the “modified FGF-21” encompass FGF-21 polypeptidescomprising one or more non-naturally encoded amino acid substitutions oradditions. In some embodiments, the “modified FGF-21” encompass FGF-21polypeptides comprising one or more non-naturally encoded amino acidsubstitutions.

In some cases, the non-naturally encoded amino acid substitution(s) maybe combined with other additions, substitutions or deletions within themodified FGF-21 polypeptide to affect other biological traits of themodified FGF-21 polypeptide relative to another FGF-21 polypeptide.

In some cases, the other additions, substitutions or deletions mayincrease the stability (including but not limited to, resistance toproteolytic degradation) of the modified FGF-21 polypeptide or increaseaffinity of the modified FGF-21 polypeptide for its receptor. In somecases, the other additions, substitutions or deletions may increase thepharmaceutical stability of the modified FGF-21 polypeptide. In somecases, the other additions, substitutions or deletions may increase thesolubility (including but not limited to, when expressed in E. coli orother host cells) of the modified FGF-21 polypeptide. In someembodiments, the other addition, substitution or deletion may modulateaffinity of the modified FGF-21 polypeptide for its receptor, bindingproteins, or associated ligand, modulate signal transduction afterbinding to its receptor, modulates circulating half-life, modulaterelease or bio-availability, facilitates purification, or improve oralter a particular route of administration. In some embodiments,modified FGF-21 polypeptides can comprise chemical or enzyme cleavagesequences, protease cleavage sequences, reactive groups,antibody-binding domains (including but not limited to, FLAG orpoly-His) or other affinity based sequences (including, but not limitedto, FLAG, poly-His, GST, etc.) or linked molecules (including, but notlimited to, biotin) that improve detection (including, but not limitedto, GFP), purification, transport through tissues or cell membranes, orother traits of the polypeptide.

Multiple polymorphisms of FGF-21 have been identified. Leucine orproline have been described at the same position in U.S. PatentPublication No. 20010012628 and U.S. Pat. No. 6,716,626. N-terminalleader or signal sequences that differ by 1 amino acid (leucine) areshown in U.S. Pat. No. 6,716,626 and U.S. Patent Publication No.20040259780. FGF-21 polypeptide variants or mutants include, but are notlimited to, those disclosed in U.S. Pat. No. 6,716,626; U.S. PatentPublication Nos. 2005/0176631, 2005/0037457, 2004/0185494, 2004/0259780,2002/0164713, and 2001/0012628; WO 01/36640; WO 03/011213; WO 03/059270;WO 04/110472; WO 05/061712; WO 05/072769; WO 05/091944; WO 05/113606; WO06/028595; WO 06/028714; WO 06/050247; WO 06/065582; WO 06/078463;WO01/018172; WO09/149171; WO10/042747; WO12/066075; WO11/154349;WO13/052311; WO13/188181, which are expressly incorporated by referencein their entireties herein.

The term “modified FGF-21 polypeptide” also includes biologically-activefragments, biologically active variants and stereoisomers of thenaturally-occurring FGF-21 as well as agonist, mimetic, and antagonistvariants of the naturally-occurring FGF-21 and polypeptide fusionsthereof. Fusions comprising additional amino acids at the aminoterminus, carboxyl terminus, or both, are encompassed by the term“modified FGF-21 polypeptide.” Exemplary fusions include, but are notlimited to, e.g., methionyl FGF-21 in which a methionine is linked tothe N-terminus of FGF-21 resulting from the recombinant expression ofthe mature form of FGF-21 lacking the leader or signal peptide orportion thereof (a methionine is linked to the N-terminus of FGF-21resulting from the recombinant expression, e.g. in E. coli), fusions forthe purpose of purification (including, but not limited to, topoly-histidine or affinity epitopes), fusions with serum albumin bindingpeptides such as PK extending (PKE) adnectin and fusions with serumproteins such as serum albumin, and fusion proteins comprising FGF-21and one or more other molecules (“fusion partner”), including but notlimited to, serum albumin, Fc domain, immunoglobulin constant region,unstructured polypeptide, and adnectin, and a fragment thereof. Any suchfragments can be prepared from the proteins by standard biochemicalmethods, or by expressing a polynucleotide encoding the fragment.

The term “modified FGF-21 polypeptide” includes polypeptides conjugatedto a polymer such as PEG and may optionally comprise one or moreadditional derivitizations of cysteine, lysine, or other residues. Forexample, the modified FGF-21 polypeptide may be conjugated to a linkeror polymer, wherein the linker or polymer may be conjugated to anon-natural amino acid in the modified FGF-21 polypeptide according tothe present disclosure, or may be conjugated to a naturally encodedamino acid utilizing techniques known in the art such as coupling tolysine or cysteine. Exemplary linkers including but are not limited to,small organic compounds, water soluble polymers of a variety of lengthssuch as poly(ethylene glycol) or polydextran, or peptide or polypeptidesof various lengths.

The term “modified FGF-21 polypeptide” also includes glycosylatedmodified FGF-21, such as but not limited to, polypeptides glycosylatedat any amino acid position, N-linked or O-linked glycosylated forms ofthe polypeptide. Variants containing single nucleotide changes are alsoconsidered as biologically active variants of FGF-21 polypeptide. Inaddition, splice variants are also included. The term “modified FGF-21polypeptide” also includes FGF-21 polypeptide heterodimers, homodimers,heteromultimers, or homomultimers of any one or more unmodified ormodified FGF-21 polypeptides or any other polypeptide, protein,carbohydrate, polymer, small molecule, linker, ligand, or otherbiologically active molecule of any type, linked by chemical means orexpressed as a fusion protein, as well as polypeptide analoguescontaining, for example, specific deletions or other modifications yetmaintain biological activity.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally encoded amino acidsinclude, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,methionine, peptides or proteins such as serum albumin, Fc domain,immunoglobulin constant region, unstructured polypeptide, adnectin, or afragment thereof, or other moieties that increase serum (in vivo)half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” is used herein to refer to groups or bonds thatnormally are formed as the result of a chemical reaction and typicallyare covalent linkages. Hydrolytically stable linkages means that thelinkages are substantially stable in water and do not react with waterat useful pH values, including but not limited to, under physiologicalconditions for an extended period of time, perhaps even indefinitely.Hydrolytically unstable or degradable linkages mean that the linkagesare degradable in water or in aqueous solutions, including for example,blood. Enzymatically unstable or degradable linkages mean that thelinkage can be degraded by one or more enzymes. As understood in theart, PEG and related polymers may include degradable linkages in thepolymer backbone or in the linker group between the polymer backbone andone or more of the terminal functional groups of the polypetidemolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide. Exemplarylinkages also include oxime linkage resulted from the reaction of acarbonyl group and aminooxy group.

Modified FGF-21 polypeptides suitable for use in the invention can begenerated using methods well known in the art. Alternatively, artrecognized modified FGF-21 polypeptides can be used. For examplemodified FGF-21 polypeptides are described in U.S. Pat. Nos. 9,079,971and 9,434,778, the contents of which are expressly incorporated hereinby reference in their entireties.

An exemplary modified FGF-21 may be a pegylated analogue of humanFGF-21. In one embodiment, the modified FGF-21 comprises the polypeptideof SEQ ID NO:1, except that an amino acid in the polypeptide issubstituted by a non-naturally encoded amino acid, wherein saidnon-naturally encoded amino acid is at a position corresponding toresidue 108 of SEQ ID NO:1.

In another embodiment, the modified FGF-21 comprises the polypeptide ofSEQ ID NO:1, except that an amino acid in the polypeptide is substitutedby a non-naturally encoded amino acid, wherein: (a) said non-naturallyencoded amino acid is at a position corresponding to residue 108 of SEQID NO:1; (b) said non-naturally encoded amino acid is linked to apolymer comprising a poly(ethylene glycol).

In another embodiment, the modified FGF-21 comprises the polypeptide ofSEQ ID NO:1 except that the amino acid at position 108 of SEQ ID NO:1 issubstituted by a non-naturally encoded amino acid, wherein: (a) saidnon-naturally encoded amino acid comprises para-acetyl phenylalanine,and (b) said non-naturally encoded amino acid is linked to a polymercomprising a poly(ethylene glycol). In one embodiment, the poly(ethyleneglycol) may have an average molecular weight of about 30 kDa.

In another embodiment, the non-naturally encoded amino acid is linked tosaid polymer through an oxime linkage.

In another embodiment, the modified FGF-21 comprises SEQ ID NO:2.

In another embodiment, the modified FGF-21 comprises SEQ ID NO:2,wherein the para-acetyl phenylalanine in SEQ ID NO:2 is linked to apolymer comprising a poly(ethylene glycol).

In another embodiment, the modified FGF-21 comprises SEQ ID NO:2,wherein the para-acetyl phenylalanine in SEQ ID NO:2 is linked to apolymer comprising a poly(ethylene glycol) having an average molecularweight of about 30 kDa. In another embodiment, the modified FGF-21 maybe BMS-986036.

Also provided is a modified FGF-21 for use in the methods describedherein. In one embodiment, a modified FGF-21 for use in a method oftreating NASH in a patient is provided, wherein the method comprisesadministering to the patient a modified FGF-21 in an amount and with afrequency sufficient to treat NASH, wherein the patient has beendetermined to have a serum Pro-C3 level greater than 10, 15 or 20 ng/ML.In another embodiment, a modified FGF-21 for use in a method ofmonitoring responsiveness of a patient having NASH to treatment with amodified FGF-21 is provided, wherein a decreased serum Pro-C3 level in ablood sample from the patient obtained during or after treatment, ascompared to a serum Pro-C3 level in a blood sample from the patientobtained prior to treatment with the modified FGF-21, indicates that thepatient is responsive to treatment with the modified FGF-21.

3. Samples and Collection

Suitable biological samples for use in the methods described hereininclude whole blood (or a fraction thereof). A biological sample can befurther fractionated, if desired, to a fraction containing particularanalytes (e.g., proteins) of interest. For example, a whole blood samplecan be fractionated into serum or into fractions containing particulartypes of proteins.

The biological samples can be obtained from a subject, e.g., a patienthaving, suspected of having, or at risk of developing NASH. Any suitablemethods for obtaining the biological samples can be employed.

In some embodiments, a protein extract may be prepared from a biologicalsample. In some embodiments, a protein extract contains the totalprotein content. Methods of protein extraction are well known in theart. See, e.g., Roe (2001) “Protein Purification Techniques: A PracticalApproach”, 2^(nd) Edition, Oxford University Press. Numerous differentand versatile kits can be used to extract proteins from bodily fluidsand tissues, and are commercially available from, for example, BioRadLaboratories (Hercules, CA), BD Biosciences Clontech (Mountain View,CA), Chemicon International, Inc. (Temecula, CA), Calbiochem (San Diego,CA), Pierce Biotechnology (Rockford, IL), and Invitrogen Corp.(Carlsbad, CA).

Methods for obtaining and/or storing samples that preserve the activityor integrity of cells in the biological sample are well known to thoseskilled in the art. For example, a biological sample can be furthercontacted with one or more additional agents such as appropriate buffersand/or inhibitors, including protease inhibitors, the agents meant topreserve or minimize changes (e.g., changes in osmolarity or pH) inprotein structure. Such inhibitors include, for example, chelators suchas ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraaceticacid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride(PMSF), aprotinin, and leupeptin. Appropriate buffers and conditions forstoring or otherwise manipulating whole cells are described in, e.g.,Pollard and Walker (1997), “Basic Cell Culture Protocols,” volume 75 ofMethods in molecular biology, Humana Press; Masters (2000) “Animal cellculture: a practical approach,” volume 232 of Practical approach series,Oxford University Press; and Jones (1996) “Human cell cultureprotocols,” volume 2 of Methods in molecular medicine, Humana Press.

A sample also can be processed to eliminate or minimize the presence ofinterfering substances. For example, a biological sample can befractionated or purified to remove one or more materials (e.g., cells)that are not of interest. Methods of fractionating or purifying abiological sample include, but are not limited to, flow cytometry,fluorescence activated cell sorting, and sedimentation.

4. Methods for Treatment

Also provided herein are methods for treating a patient having NASH byadministering to the patient a modified Fibroblast growth factor 21(FGF-21) if the patient has been determined to have a particular serumthreshold level of Pro-C3 (e.g., greater than about 10 ng/ML, 11 ng/ML,12 ng/ML, 13 ng/ML, 14 ng/ML, 15 ng/ML, 16 ng/ML, 17 ng/ML, 18 ng/ML, 19ng/ML, 20 ng/ML, 22 ng/ML, 23 ng/ML).

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic measures described herein. The methods of treatment employadministration to a patient (such as a human) the combination disclosedherein in order to cure, delay, reduce the severity of, or ameliorateone or more symptoms of the disease or disorder or recurring disease ordisorder, or in order to prolong the survival of a patient beyond thatexpected in the absence of such treatment.

The terms “effective amount” or “therapeutically effective amount” areused interchangeably and refer to an amount of formulation effective toalleviate or ameliorate one or more symptom(s) of the disease ordisorder (e.g., NASH) or to prolong the survival of the patient beingtreated. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein. Therapeutically effectivedosages may be determined by using in vitro and in vivo methods.

In one embodiment, a method for treating a patient having NASH isprovided comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFGF-21 in an amount and with a frequency sufficient to treat NASH, ifthe serum Pro-C3 level is greater than about 10 ng/ML prior toadministration to the patient of the modified FGF-21.

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH.

In another embodiment, a method for treating a patient with NASH withmodified FGF-21 is provided, wherein the patient has a serum Pro-C3level greater than 10 ng/ML prior to treatment, wherein the methodcomprises administering to the patient a modified FGF-21 in an amountand with a frequency sufficient to treat NASH.

In another embodiment, a method for treating a patient having NASH isprovided, wherein the method comprises: (1) obtaining or having obtaineda blood sample from the patient, (2) determining or having determined aserum Pro-C3 level in the blood sample that is greater than 10 ng/ML,and (3) administering to the patient a modified FGF-21 in an amount andwith a frequency sufficient to treat NASH after a serum Pro-C3 level hasbeen determined.

In another embodiment a method for treating a patient having NASH isprovided, comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFGF-21 in an amount and with a frequency sufficient to treat NASH, ifthe serum Pro-C3 level is greater than about 10 ng/ML, wherein themodified FGF-21 comprises the polypeptide of SEQ ID NO:1, except that anamino acid in the polypeptide is substituted by a non-naturally encodedamino acid, wherein: (a) said non-naturally encoded amino acid is at aposition corresponding to residue 108 of SEQ ID NO:1; (b) saidnon-naturally encoded amino acid comprises para-acetyl phenylalaninelinked to a polymer comprising a poly(ethylene glycol).

In another embodiment a method for treating a patient having NASH isprovided, comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFGF-21 in an amount and with a frequency sufficient to treat NASH, ifthe serum Pro-C3 level is greater than about 10 ng/ML, wherein themodified FGF-21 comprises the polypeptide of SEQ ID NO:1 except that theamino acid at position 108 of SEQ ID NO:1 is substituted by anon-naturally encoded amino acid, wherein: (a) said non-naturallyencoded amino acid comprises para-acetyl phenylalanine, and (b) saidnon-naturally encoded amino acid is linked to a polymer comprising apoly(ethylene glycol) having an average molecular weight of about 30kDa.

In another embodiment, a method for treating a patient having NASH isprovided, comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFGF-21 in an amount and with a frequency sufficient to treat NASH, ifthe serum Pro-C3 level is greater than about 10 ng/ML, wherein themodified FGF-21 consists of or comprises SEQ ID NO:2.

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH, wherein the modified FGF-21 comprises the polypeptide of SEQ IDNO:1, except that an amino acid in the polypeptide is substituted by anon-naturally encoded amino acid, wherein: (a) said non-naturallyencoded amino acid is at a position corresponding to residue 108 of SEQID NO:1; (b) said non-naturally encoded amino acid comprises para-acetylphenylalanine linked to a polymer comprising a poly(ethylene glycol).

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH, wherein the modified FGF-21 comprises the polypeptide of SEQ IDNO:1 except that the amino acid at position 108 of SEQ ID NO:1 issubstituted by a non-naturally encoded amino acid, wherein: (a) saidnon-naturally encoded amino acid comprises para-acetyl phenylalanine,and (b) said non-naturally encoded amino acid is linked to a polymercomprising a poly(ethylene glycol) having an average molecular weight ofabout 30 kDa.

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH, wherein the modified FGF-21 consists of or comprises SEQ ID NO:2.

In another embodiment, a method for treating a patient having NASH whohas been determined to have a serum Pro-C3 level greater than about 10ng/ML is provided, the method comprising administering to the patient amodified FGF-21 in an amount and with a frequency sufficient to treatNASH, wherein the modified FGF-21 consists of or comprises SEQ ID NO:2,wherein the non-naturally encoded amino acid in SEQ ID NO: 2 is linkedto a polymer comprising a poly(ethylene glycol) having an averagemolecular weight of about 30 kDa.

In another embodiment, a modified FGF-21 for use in a method of treatingNASH in a patient is provided, wherein the method comprisesadministering to the patient a modified FGF-21 in an amount and with afrequency sufficient to treat NASH, wherein the patient has beendetermined to have a serum Pro-C3 level greater than 10, 15 or 20 ng/ML.

The modified FGF-21 can be administered to a subject, e.g., a humanpatient, using a variety of methods that depend, in part, on the routeof administration. The route can be, e.g., intravenous injection orinfusion (IV), subcutaneous injection (SC), intraperitoneal (IP)injection, or intramuscular injection.

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the patient. See, e.g., U.S. patentpublication no. 20080241223; U.S. Pat. Nos. 5,501,856; 4,863,457; and3,710,795; and European patent nos. EP488401 and EP430539, thedisclosures of each of which are incorporated herein by reference intheir entirety. The composition can be delivered to the patient by wayof an implantable device based on, e.g., diffusive, erodible orconvective systems, e.g., osmotic pumps, biodegradable implants,electrodiffusion systems, electroosmosis systems, vapor pressure pumps,electrolytic pumps, effervescent pumps, piezoelectric pumps,erosion-based systems, or electromechanical systems.

A suitable dose of a modified FGF-21 which is capable of treating NASHin a patient, can depend on a variety of factors including, e.g., theage, sex, and weight of a patient to be treated. Other factors affectingthe dose administered to the patient include, e.g., the severity of thedisease. Other factors can include, e.g., other medical disordersconcurrently or previously affecting the patient, the general health ofthe patient, the genetic disposition of the patient, diet, time ofadministration, rate of excretion, drug combination, and any otheradditional therapeutics that are administered to the patient. It shouldalso be understood that a specific dosage and treatment regimen for anyparticular patient will depend upon the judgment of the treating medicalpractitioner (e.g., doctor or nurse).

In one embodiment, the modified FGF-21 is administered at a fixed dose.As used herein, the terms “fixed dose”, “flat dose”, and “flat-fixeddose” are used interchangeably and refer to a dose that is administeredto a patient without regard for the weight or body surface area (BSA) ofthe patient. The fixed or flat dose is therefore not provided as a mg/kgdose, but rather as an absolute amount of the agent.

In one embodiment, the modified FGF-21 is administered at a fixed weeklydose. In another embodiment, the modified FGF-21 is administered at adose of about 10 mg once weekly. In another embodiment, the modifiedFGF-21 is administered at a dose of about 11 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 12 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 13 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 14 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 15 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 16 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 17 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 18 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 19 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 20 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 21 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 22 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 23 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 24 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 25 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 26 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 27 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a dose of about 28 mg once weekly. In another embodiment, themodified FGF-21 is administered at a dose of about 29 mg once weekly. Inanother embodiment, the modified FGF-21 is administered at a dose ofabout 30 mg once weekly. In another embodiment, the modified FGF-21 isadministered at a dose of about 35 mg once weekly. In anotherembodiment, the modified FGF-21 is administered at a dose of about 40 mgonce weekly. In another embodiment, the modified FGF-21 is administeredat a fixed daily dose. In some embodiments, the modified FGF-21 isadministered at a dose of about 5 mg once daily, about 6 mg once daily,about 7 mg once daily, about 8 mg once daily, about 9 mg once daily,about 10 mg once daily, about 11 mg once daily, about 12 mg once daily,about 13 mg once daily, about 14 mg once daily, about 15 mg once daily,about 16 mg once daily, about 17 mg once daily, about 18 mg once daily,about 19 mg once daily, about 20 mg once daily, about 25 mg once daily,or about 30 mg once daily. In one embodiment, the modified FGF-21 isadministered at a dose of about 10 mg once daily. In another embodiment,the modified FGF-21 is administered at a dose of about 15 mg once daily.In another embodiment, the modified FGF-21 is administered at a dose ofabout 20 mg once daily.

In some embodiments, the modified FGF-21 is administered at a fixed dosefor 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months,8 months, 9 months, 10 months, 11 months, or 12 months.

A pharmaceutical composition can include a therapeutically effectiveamount of a modified FGF-21 (e.g., such as BMS-986036). Such effectiveamounts can be readily determined by one of ordinary skill in the artbased, in part, on the effect of the modified FGF-21 or thecombinatorial effect of the modified FGF-21 and one or more additionalactive agents, if more than one agent is used. A therapeuticallyeffective amount of a modified FGF-21 can also vary according to factorssuch as the disease state, age, sex, and weight of the individual, andthe ability of the modified FGF-21 (and one or more additional activeagents) to elicit a desired response in the individual, e.g.,amelioration of at least one condition parameter, e.g., amelioration ofat least one symptom of NASH. For example, a therapeutically effectiveamount of a modified FGF-21 can inhibit (lessen the severity of oreliminate the occurrence of) of any one of the symptoms of NASH. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

Toxicity and therapeutic efficacy of a modified FGF-21 can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals. These procedures can be used, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compositions that exhibit hightherapeutic indices are preferred. While compositions that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue and tominimize potential damage to normal cells and, thereby, reduce sideeffects.

5. Methods of Monitoring Responsiveness

Also provided are methods for monitoring responsiveness of a patienthaving NASH to treatment with a modified FGF-21, the method comprising:determining the serum Pro-C3 level in a blood sample from the patientobtained during or after treatment, wherein: a decreased serum Pro-C3level in the blood sample from the patient obtained during or aftertreatment, as compared to the serum Pro-C3 level in a blood sample fromthe patient obtained prior to treatment with the modified FGF-21,indicates that the patient is responsive to treatment with the modifiedFGF-21.

In another embodiment, a method for monitoring responsiveness of apatient having NASH to treatment with a modified FGF-21 is provided, themethod comprising: determining the serum Pro-C3 level in a blood samplefrom the patient obtained during or after treatment, wherein: adecreased serum Pro-C3 level in the blood sample from the patientobtained during or after treatment, as compared to the serum Pro-C3level in a blood sample from the patient obtained prior to treatmentwith the modified FGF-21, indicates that the patient is responsive totreatment with the modified FGF-21, wherein the modified FGF-21comprises the polypeptide of SEQ ID NO:1, except that an amino acid inthe polypeptide is substituted by a non-naturally encoded amino acid,wherein: (a) said non-naturally encoded amino acid is at a positioncorresponding to residue 108 of SEQ ID NO:1; (b) said non-naturallyencoded amino acid comprises para-acetyl phenylalanine linked to apolymer comprising a poly(ethylene glycol)

In another embodiment, a method for monitoring responsiveness of apatient having NASH to treatment with a modified FGF-21 is provided, themethod comprising: determining the serum Pro-C3 level in a blood samplefrom the patient obtained during or after treatment, wherein: adecreased serum Pro-C3 level in the blood sample from the patientobtained during or after treatment, as compared to the serum Pro-C3level in a blood sample from the patient obtained prior to treatmentwith the modified FGF-21, indicates that the patient is responsive totreatment with the modified FGF-21, wherein the modified FGF-21comprises the polypeptide of SEQ ID NO:1 except that the amino acid atposition 108 of SEQ ID NO:1 is substituted by a non-naturally encodedamino acid, wherein: (a) said non-naturally encoded amino acid comprisespara-acetyl phenylalanine, and (b) said non-naturally encoded amino acidis linked to a polymer comprising a poly(ethylene glycol) having anaverage molecular weight of about 30 kDa.

In another embodiment, a method for monitoring responsiveness of apatient having NASH to treatment with a modified FGF-21 is provided, themethod comprising: determining the serum Pro-C3 level in a blood samplefrom the patient obtained during or after treatment, wherein: adecreased serum Pro-C3 level in the blood sample from the patientobtained during or after treatment, as compared to the serum Pro-C3level in a blood sample from the patient obtained prior to treatmentwith the modified FGF-21, indicates that the patient is responsive totreatment with the modified FGF-21, wherein the modified FGF-21 consistsof or comprises SEQ ID NO:2.

In another embodiment, a modified FGF-21 for use in a method ofmonitoring responsiveness of a patient having NASH to treatment with amodified FGF-21 is provided, wherein a decreased serum Pro-C3 level in ablood sample from the patient obtained during or after treatment, ascompared to a serum Pro-C3 level in a blood sample from the patientobtained prior to treatment with the modified FGF-21, indicates that thepatient is responsive to treatment with the modified FGF-21.

In one embodiment, a decrease in serum levels of Pro-C3 by at least 5(e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70) %following administration of the modified FGF-21, indicates that thepatient is responsive to treatment with the modified FGF-21.

In another embodiment, a decrease in serum levels of Pro-C3 to within 90(e.g., 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30) % of theBaseline serum level of Pro-C3 following administration of one or moredoses of the modified FGF-21, indicates that the patient is responsiveto treatment with the modified FGF-21.

6. Methods for Identifying Responders

Also provided are methods for identifying responder of a patient havingNASH to treatment with a modified FGF-21. In one embodiment, the methodcomprises: determining the patient has a serum Pro-C3 level greater thanabout 10 ng/ML in a blood sample obtained prior to treatment, andidentifying the patient as a responder to the treatment with themodified FGF-21, wherein the modified FGF-21 comprises the polypeptideof SEQ ID NO:1 except that the amino acid at position 108 of SEQ ID NO:1is substituted by a non-naturally encoded amino acid, wherein: (a) saidnon-naturally encoded amino acid comprises para-acetyl phenylalanine,and (b) said non-naturally encoded amino acid is linked to a polymercomprising a poly(ethylene glycol) having an average molecular weight ofabout 30 kDa. In another embodiment, a method of identifying a patienthaving NASH that is suitable for treatment with a modified FGF-21 isprovided, wherein the method comprises determining a serum Pro-C3 levelin a blood sample from the patient using an in vitro assay, wherein theserum Pro-C3 level in the blood sample is greater than 10, 15 or 20ng/ML. In some embodiments, the method comprises determining the patienthas a serum Pro-C3 level greater than about 11 ng/ML, 12 ng/ML, 13ng/ML, 14 ng/ML, 15 ng/ML, 16 ng/ML, 17 ng/ML, 18 ng/ML, 19 ng/ML, 20ng/ML, 21 ng/ML, or 22 ng/ML, in a blood sample obtained from thepatient prior to treatment.

7. Additional Agents/Therapies

In some embodiments, the modified FGF-21 can be administered to apatient as a monotherapy. Alternatively, as described above, themodified FGF-21 can be administered to a patient as a combinationtherapy with another treatment. For example, the combination therapy caninclude administering to the patient (e.g., a human patient) one or moreadditional agents that provide a therapeutic benefit to the patient whohas NASH. In one embodiment, the modified FGF-21 is administered firstin time and the one or more additional active agents are administeredsecond in time. In some embodiments, the one or more additional activeagents are administered first in time and the modified FGF-21 isadministered second in time.

In some embodiments, the one or more additional therapeutic agents maybe selected from anti-fibrotic agents, N-cadherin antagonist, anti-Ncadherin antibody, small molecule N-cadherin antagonist, antagonisticN-cadherin fragment, anti-inflammatory agents, hepatoprotective agentssuppressing renin-angiotensin system (RAS) system, probiotics, andpolyunsaturated fatty acids (PUFAs). In some embodiments, theanti-fibrotic agent may be selected from nintedanib, Pirfenidone, LPA1antagonists, LPA1 receptor antagonists, GLP1 analog, tralokinumab(IL-13, AstraZeneca), vismodegib (hedgehog antagonist, Roche), PRM-151(pentraxin-2, TGF beta-1, Promedior), SAR-156597 (bispecific MabIL-4&IL-13, Sanofi), simtuzumab (anti-lysyl oxidase-like 2 (anti-LOXL2)antibody, Gilead), CKD-942, PTL-202 (PDE inh./pentoxifylline/NAC oralcontrol. release, Pacific Ther.), omipalisib (oral PI3K/mTOR inhibitor,GSK), IW-001 (oral sol. bovine type V collagen mod., ImmuneWorks),STX-100 (integrin alpha V/beta-6 ant, Stromedix/Biogen), Actimmune (IFNgamma), PC-SOD (midismase; inhaled, LTT Bio-Pharma/CKD Pharm),lebrikizumab (anti-IL-13 SC humanized mAb, Roche), AQX-1125 (SHIP1activator, Aquinox), CC-539 (JNK inhibitor, Celgene), FG-3019(FibroGen), and SAR-100842 (Sanofi). In some embodiments, thehepatoprotective agent may be ursodeoxycholic acid (UDCA) or obeticholicacid (OCA or INT-747, Intercept).

8. Outcomes

Patients treated according to the methods disclosed herein preferablyexperience improvement in at least one sign of NASH.

In one embodiment, the treatment methods described herein result in adecrease in Pro-C3 levels (e.g., at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, or 70%) compared to pre-treatment Pro-C3 levels. Inanother embodiment, the treatment methods described herein result in adecrease in Pro-C3 levels (e.g., by at least about 1.5-fold, 2-fold,2.5-fold, or 3-fold compared to pre-treatment Pro-C3 levels).

In another embodiment, the treatment methods described herein produce ashift toward normal levels of Pro-C3 (e.g., to within 50 (e.g., 49, 48,47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) % above the normal serum level ofPro-C3).

In another embodiment, the treatment methods described herein result ina reduction in liver stiffness in a patient, as assessed by magneticresonance elastography (MRE). For example, in one embodiment, treatmentwith a modified FGF-21 results in a reduction in liver stiffness in thepatient compared to the patient's liver stiffness prior to treatment,wherein liver stiffness is assessed by MRE. In another embodiment,treatment with a modified FGF-21 results in a 15% or greater (e.g., 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 50%, 55%, 60%, 65%, 70%, 75% or greater) reduction in liverstiffness in the patient compared to the patient's liver stiffness priorto treatment, wherein liver stiffness is assessed by MRE.

In another embodiment, the treatment methods described herein result ina reduction in hepatic fat fraction in a patient. For example, in oneembodiment, treatment with a modified FGF-21 results in a reduction inhepatic fat fraction in the patient compared to the patient's hepaticfat fraction prior to treatment, wherein hepatic fat fraction is asassessed by magnetic resonance imaging-estimated proton density fatfraction (MRI-PDFF). In another embodiment, treatment with the modifiedFGF-21 results in a 30% or greater (e.g., 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75% orgreater) reduction in hepatic fat fraction in the patient compared tothe patient's hepatic fat fraction prior to treatment, wherein hepaticfat fraction is assessed by MRI-PDFF. In another embodiment, thepatient's hepatic fat fraction is decreased by at least 5 (e.g., atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70) % followingadministration of the modified FGF-21 to the patient, compared to thepatient's hepatic fat fraction prior to treatment, as assessed byMRI-PDFF. In another embodiment, the patient's hepatic fat fraction isdecreased by at least 20% following administration of the modifiedFGF-21 to the patient, compared to the patient's hepatic fat fractionprior to treatment, as assessed by MRI-PDFF. In another embodiment, thepatient's hepatic fat fraction is decreased by at least 30% followingadministration of the modified FGF-21 to the patient, compared to thepatient's hepatic fat fraction prior to treatment, as assessed byMRI-PDFF. In another embodiment, the patient's hepatic fat fraction isdecreased to within 90 (e.g., 85, 80, 75, 70, 65, 60, 55, 50, 45, 40,35, or 30) % of the patient's pre-treatment (prior to treatment)baseline serum level of hepatic fat fraction following administration ofthe modified FGF-21.

In another embodiment, the treatment methods described herein result ina reduction in live stiffness, a reduction in hepatic fat fraction,and/or a decrease in serum Pro-C3 levels in a patient. In anotherembodiment, treatment with a modified FGF-21 results in a reduction inliver stiffness in the patient compared to the patient's liver stiffnessprior to treatment, and a decrease in serum Pro-C3 levels in the patientcompared to the patient's serum Pro-C3 levels prior to treatment,wherein liver stiffness is assessed by MRE, and wherein the patient hasbeen determined to have a serum Pro-C3 level greater than 10, 15 or 20ng/ML prior to treatment with the modified FGF-21. In anotherembodiment, the treatment methods described herein produces at least onetherapeutic effect selected from the group consisting of a reduction orcessation in fatigue, malaise, weight loss, and/or right upper quadrantabdominal discomfort in the patient.

9. Kits

Also provided are kits comprising various reagents and materials usefulfor carrying out the methods described herein. The procedures formeasuring, diagnosing, evaluating, and/or assessing described herein maybe performed by diagnostic laboratories, experimental laboratories, orindividual practitioners. The invention provides kits which can be usedin any or all of these settings. In some embodiments, the kits describedherein comprise materials and reagents for, among other things,characterizing or processing biological samples (e.g., blood), measuringPro-C3 biomarker levels (e.g., protein or nucleic acid levels),monitoring treatment response in a patient according to the methodsprovided herein. In certain embodiments, an inventive kit comprises atleast one or more reagents that specifically detect serum protein levelsof Pro-C3 and, optionally, instructions for using the kit.

In some embodiments, the kits may include suitable control samples(e.g., biological samples from normal healthy individuals or a solutioncomprising a known, control amount of a particular analyte of interest,such as Pro-C3). In some embodiments, kits of the invention may includeinstructions for using the kit according to one or more methodsdescribed herein and may comprise instructions for processing thebiological sample (e.g., blood) obtained from the patient and/or forperforming the test or instructions for interpreting the results.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, orprocess step or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

The following example is intended to illustrate, not limit, theinvention.

EXAMPLES Example 1: A Randomized, Double-Blind, Placebo-Controlled,Parallel Group, Multiple Dose Study to Evaluate the Safety,Pharmacokinetics and Pharmacodynamic Effects of BMS-986036 in Adultswith Nonalcoholic Steatohepatitis (NASH)

The objectives of this study were to assess the effect of 16 weeks ofdaily or weekly doses of BMS-986036 (a modified FGF21 comprising theamino acid sequence of SEQ ID NO:2, in which the pAF is linked to a 30kD PEG) on safety, tolerability and change in hepatic fat fraction (%)by MRI in patients with NASH and to assess the pharmacokinetics andimmunogenicity of BMS-986036 in patients with NASH. The objectives alsoincluded assessing the effect of daily or weekly doses of BMS-986036 onLiver stiffness by MRE at 16 weeks, Body composition by dual-energyX-ray absorptiometry (DXA), BMS-986036 urinary concentration, Bodyweight and waist circumference, ALT (alanine aminotransferase) and AST(aspartate aminotransferase) levels, Glucose homeostasis and insulinsensitivity, Fasting lipids, Bone homeostasis, exploratory biomarkersassociated with the risk of disease progression and complications, andCalculated indices related to NASH.

A. Methods

Subjects underwent screening evaluations to determine eligibility within42 days prior to randomization. Eligible subjects were randomized to oneof three parallel treatment groups and self-administered double-blindtreatment, once daily or once weekly for 16 weeks. Included in the studywere male and female subjects aged 21 to 75 years with a BMI of ≥25kg/m² with a liver biopsy performed within 1 year of Screening (orbetween Screening and Lead-in) with documented results of NASH with NASHCRN fibrosis stage 1-3 and a hepatic fat fraction (%)≥10% by magneticresonance imaging-proton density fat fraction (MRI-PDFF). Patients wereexcluded if they had evidence of a medical condition contributing tochronic liver disease other than NASH, evidence of cirrhosis,decompensated liver disease, glycated haemoglobin (Hb_(A1c))≥9·5%,recent drug or alcohol abuse or significant alcohol consumption, bonetrauma, fracture, or bone surgery within 8 weeks of screening, or anyclinically significant deviation from normal in physical examination orclinical laboratory determinations beyond what is consistent with thetarget patient population.

Eligible patients were randomised (1:1:1) on Day 1 using the InteractiveVoice Response System (IVRS) to one of the following groups: BMS-98603610 mg QD, BMS-986036 20 mg QW, or placebo QD. Patients were stratifiedby diagnosis of T2DM status based on current American DiabetesAssociation criteria (see American Diabetes Association, “Classificationand diagnosis of diabetes”, Sec. 2. Diabetes Care 2015; 38(Suppl 1):S8). Blinded treatments were supplied in numbered kits foradministration in an outpatient setting. At all study visits when studydrugs were dispensed, each patient was randomly assigned a kit number bythe IVRS. Kit numbers corresponded to the numbers printed on thepackages and kits containing study drug. Each kit contained 8 vials tosupport one week of dosing. 2 vials were designated for Day 1 and 1 vialwas provided for each of Days 2-7.

Clinic visits were scheduled approximately every 2 weeks initially, andthen monthly, to collect safety, PK, and PD measures (Table 1).

TABLE 1 Study Design Schematic Visit Days D −42 D −7 D 1 D 15 D 29 D 43D 57 D 86 D 112 D 142 D 292 Screening Lead-in On treatment Follow-up 5weeks 1 week 4 months Placebo Treatment A: 10 mg QD Treatment B: 20 mgQW Treatment C: Placebo QD QD = Once daily; QW = Once weekly; D = Day

Prior to randomisation, eligible patients completed a 7-day study skillslead-in period consisting of daily placebo injections (Day −7 to Day−1). Injections were self-administered subcutaneously in the abdomen andpatients were trained to rotate injection sites relative to theumbilicus. After randomisation on Day 1, patients self-administereddouble-blind treatment subcutaneously once daily for 16 weeks as setforth in Table 2. For all treatments, on Day 1 of each treatment week,two 1-mL injections were administered concurrently and on Days 2 to 7,one 1-mL injection was administered. For the BMS-986036 10 mg QD group,the two injections on Day 1 consisted of one active dose and one placeboto maintain the blind between daily and weekly treatment groups. For theBMS-986036 20 mg QW group, on Days 2-7, the daily injection was placeboto maintain the blind between daily and weekly treatment groups.

TABLE 2 Treatment Administration Number and volume of injectionsSolution Day 1 of each Days 2-7 of each Treatment strength treatmentweek treatment week Lead-in Placebo QD N/A 2 × 1 mL 1 × 1 mL, dailyphase A BMS-986036 10 2 × 1 mL 1 × 1 mL, daily 10 mg QD mg/mL BBMS-986036 10 2 × 1 mL 1 × 1 mL, daily 20 mg QW mg/mL C Placebo QW N/A 2× 1 mL 1 × 1 mL, daily N/A = not applicable. QD = once daily. QW = onceweekly.

On-treatment clinic visits were scheduled for Days 1, 15, 29, 43, 57, 86and 112. Post-study follow-up visits were scheduled for Days 142 and292. Physical examinations, vital sign measurements, and clinicallaboratory evaluations were performed at screening, at each treatmentvisit, and at the Day 142 follow-up visit. Twelve-leadelectrocardiograms (ECGs) were performed at screening, Day 1, Day 112,and at follow-up. Due to the association of FGF21 and bone loss observedin mouse models (see, e.g., Wei W, et al. Proc Natl Acad Sci USA 2012;109(8): 3143-8), dual emission X-ray absorptiometry (DXA) was performedto monitor bone mineral density (BMD) and body composition at screening,end of treatment, and 6 months after the end of treatment. MRI-PDFF wasperformed at screening, and on Day 57 and the end of treatment. At asubset of facilities with the appropriate hardware and software,magnetic resonance elastography (MRE) was conducted at screening and atthe end of treatment. Fasting lipids (LabCorp, Burlington, NC),adiponectin (Myriad RBM, Austin, TX), and insulin (LabCorp) levels wereassessed on Days 1, 29, 57, 86, 112, and 142. PRO-C3 levels wereassessed on Days 1, 57, and 112 (Nordic Bioscience, Herlev, Denmark).Adverse event (AE) data were collected at every time point throughoutthe study and at follow-up visits. Patients were monitored for injectionsite reactions from Day −7 through the end of the post-study follow-upvisits. The Draize scale for erythema and oedema was used as a guide forreporting injection site AEs (see Haschek W, et al. “Evaluation ofCutaneous Toxicity” In: Fundamentals of Toxicologic Pathology. Seconded. Academic Press; 2009. p. 156). Immunogenicity to study drug andendogenous FGF21 was assessed pre-dose and on Days 15, 29, 57, 86, 112,142, and 292. In addition, immunogenicity assessments were also doneapproximately every 6-8 weeks between Days 142 and 292 or up to 12months after Day 142.

Primary endpoints were safety, tolerability, and absolute change inhepatic fat fraction by MRI-PDFF in NASH patients treated with 16 weeksof QD or QW doses of BMS-986036. Secondary endpoints includedpharmacokinetics and immunogenicity. Exploratory endpoints includedchange in liver stiffness as measured by MRE at 16 weeks, bodycomposition by DXA, alanine aminotransferase (ALT) and aspartateaminotransferase (AST) levels, adiponectin, fasting lipids, and PRO-C3.Post hoc endpoints included relative improvement in hepatic fat fractionmeasured by MRI-PDFF, liver stiffness measured by MRE, and PRO-C3.

For the primary endpoint, with 27 patients per treatment group withpost-baseline measurements, it was estimated that there was 82% power todetect a difference of 5% in mean change from baseline at Week 16 inhepatic fat fraction between each of the 2 doses of BMS-986036 andplacebo at a one-sided significance level of 0-05. These calculationsassumed that the hepatic fat fraction change from baseline is normallydistributed with a standard deviation of no greater than 7% as estimatedfrom data reported in a similar population (see Le T A, et al.,Hepatology 2012; 56(3): 922-32 and Patel N S, et al., Clin GastroenterolHepatol 2015; 13(3): 561-8. To allow for about 10% of patients todropout, it was planned to randomise approximately 30 patients pertreatment group.

To evaluate the primary endpoint, a longitudinal repeated measuresanalysis was used to analyse the change in hepatic fat fraction at Week16 from baseline in treated patients with both a baseline and at leastone post-baseline measurement. The model included treatment group, weekand treatment-by-week interactions as main effects and baseline hepaticfat fraction (%) and baseline diabetic status as covariates. Anunstructured covariance matrix was used to represent the correlation ofthe repeated measures within each subject. The model provided pointestimates, standard errors and 2-sided 90% confidence intervals for meanchange from baseline within and between treatments. For exploratoryendpoints, descriptive statistics are provided by treatment and studyday. Post hoc analyses were conducted using Fisher's Exact test and notadjusted for multiple comparisons. All recorded adverse events werelisted and tabulated by system organ class, preferred term, andtreatment group. Any significant physical examination findings andclinical laboratory results were listed. A pre-specified interimanalysis was conducted after approximately 60 patients completed 8 weeksof treatment. If the mean change from baseline in hepatic fat fractioncompared with placebo was at least −4-5%, then enrolment was stopped.Analyses consisted of summaries of available data without revealing thetreatment assignment of individual patients. The results of the analysiswere reviewed by a pre-specified panel of personnel and not revealed toother study personnel. All patients who were randomized and receivedstudy drug were included in the primary analyses.

Ctrough concentrations of BMS-986036 (Total and C-Terminal intact) werederived from serum concentration versus time data. MRE was performed atspecific time points to evaluate potential changes in liver stiffness.DXA was performed at specific time points to evaluate potential changesin body composition. Blood and urine were collected to for exploratorybiomarker analysis, including Pro-C3.

Safety assessments included adverse events (AEs), serious adverse events(SAEs), and laboratory abnormalities and were based on medical review ofAE reports and the results of vital sign measurements, ECG, physicalexaminations, and clinical laboratory tests.

B. Results

184 overweight or obese patients with NASH were enrolled in the studyand 80 patients entered the lead-in phase. Of the 80 patients whoentered the lead-in phase, seventy-five subjects were randomized andtreated with BMS-986036 10 mg daily (25 subjects), BMS-986036 20 mgweekly (24 subjects), or placebo daily (26 subjects), as shown in FIG. 1. The planned sample size was 30 patients per group. However, enrolmentended early due to a significant effect of BMS-986036 observed for theprimary endpoint during the pre-planned interim analysis at treatmentWeek 8. A total of 71 of 75 patients (95%) completed the 16-weektreatment period.

Baseline demographics and disease characteristics were generallycomparable between treatment groups, as shown below in Table 3. Overall,96% (72/75) of patients were white, 64% (48/75) were women, 37% (28/75)had T2DM, and 20% (15/75) had stage 3 fibrosis, as assessed by NASH CRNcriteria. Across patient groups, baseline mean hepatic fat fractionmeasured by MRI-PDFF ranged from 18% to 21%, baseline mean liverstiffness assessed by MRE ranged from 3.1 to 3.5 kPa, and baseline meanNAS ranged from 4-0 to 4-4.

TABLE 3 Baseline Demographics and Disease Characteristics BMS-986036Placebo 10 mg QD 20 mg QW (n = 26) (n = 25) (n = 24) PatientCharacteristics Age, years, mean (SD) 47 (12) 52 (10) 52 (12) Male, n(%) 10 (39) 10 (40)  7 (29) Race, White, n (%) 25 (96) 24 (96) 23 (96)BMI, kg/m², mean (SD) 37 (7)  34 (4)  35 (6)  Disease CharacteristicsT2DM, n (%) 11 (42)  9 (36)  8 (33) NAFLD activity score 4.0 (1)   4.4(1)   4.4 (1)*  NASH CRN Fibrosis, n ( %) Stage 1 17 (65) 10 (40) 13(54) Stage 2  8 (31)  6 (24)  6 (25) Stage 3 1 (4)  9 (36)  5 (21)Liver, mean (SD) Hepatic fat fraction, % 21 (7)  18 (7)  20 (6)  Liverstiffness, kPa 3.1 (1.2) 3.5 (1.4) 3.4 (1.0) ALT, U/L 80 (51) 66 (37) 70(33) AST, U/L 58 (49) 48 (23) 52 (22) PRO-C3, ng/ml 19 (12) 19 (15) 23(15) Metabolic, mean (SD) Triglycerides, mg/dL 171 (75)*  208 (110)* 187(55)^(†) LDL cholesterol, mg/dL 128 (55)  129 (38)  120 (36)  HDLcholesterol, mg/dL 50 (11) 47 (10) 45 (12) HbA_(1c), % 6.0 (0.9) 6.1(0.9) 6.2 (1.1) ALT = alanine aminotransferase. AST = aspartateaminotransferase. BMI = body mass index. HbAlc = glycated haemoglobin.HDL = high density lipoprotein. LDL = low density lipoprotein. MRI-PDFF= magnetic resonance imaging-proton density fat-fraction. NASH CRN =Non-Alcoholic Steatohepatitis Clinical Research Network. NAFLD =non-alcoholic fatty liver disease. PRO-C3 = N-terminal type III collagenpropeptide. T2DM = type-2 diabetes mellitus. QD = once daily. QW = onceweekly. *n = 23; ^(†)n = 20.

Hepatic Fat Fraction: After 16 weeks of treatment, mean hepatic fatfraction was significantly reduced in patients who received either 10 mgQD BMS-986036 (p=0.0004) or 20 mg QW BMS-986036 (p=0.008), compared withplacebo (see FIG. 2A). Mean absolute reductions in hepatic fat fractionat Week 16 were −6.8% for the 10 mg QD dose panel (P=0.0004), −5.2% forthe 20 mg QW dose panel (P=0.008), and −1.3% for the placebo panel. Meanabsolute changes in hepatic fat fraction at Week 8 were −8.4%, −6.5%,and −1.3% for the 10 mg QD regimen, the 20 mg QW regimen, and theplacebo regimen, respectively. A total of 68 subjects had MRI-PDFF dataat both Baseline and Week 16. FIG. 2B shows the MRI-PDFF measurements ofa patient who experienced a reduction in hepatic fat fraction followingBMS-986036 treatment. Post hoc analyses showed that significantly morepatients treated with BMS-986036 10 mg QD (p=0.03) or 20 mg QW (p=0.02)compared with placebo-treated patients had ≥30% relative reduction inhepatic fat fraction (FIG. 2C). A greater proportion of patients treatedwith BMS-986036 10 mg QD or 20 mg QW compared to placebo had ≥20% or≥10% relative reductions in hepatic fat fraction. Table 4 presentspercentage of patients with >=30% relative reduction from Baseline toWeek 16 for hepatic fat fraction (56.5%, 54.5% and 24.0% for the 10 mgQD regimen, the 20 mg QW regimen, and the placebo regimen,respectively).

TABLE 4 Change from Baseline at Week 16 - Hepatic Fat Fraction Number(%) of Subjects Percent Change from BMS 10 mg daily BMS 20 mg weeklyPlacebo daily Baseline at Week 16 N = 25 N = 24 N = 26 HEPATIC FATFRACTION 13/23 (56.5) 12/22 (54.5) 6/25 (24.0) >=30% IMPROVEMENT (0.395,0.735) (0.370, 0.720) (0.099, 0.380) 90% CI ODDS RATIO (90% CI) 4.12(1.46, 11.59) 3.80 (1.34, 10.79) P-VALUE (ONE-SIDED) 0.0219 0.0318 1.Baseline is defined as last non-missing result with a collectiondate-time less than the date-time of the first active dose of studymedication. 2. Fisher exact test is conducted for this analysis.

PK: For each BMS-986036 treatment group of BMS-986036 10 mg daily andBMS-986036 20 mg weekly, the data demonstrated that steady stateconcentrations were achieved by the first week of treatment forC-terminal intact BMS-986036 and by the fourth week for Total BMS-986036groups. Ctrough remained constant during the treatment period at 10 mgQD and 20 mg QW dosing group.

MRE: Liver Stiffness: MRE was conducted to assess Liver Stiffness at thesubset of imaging facilities where appropriate hardware and software wasavailable. Therefore, the sample size for the liver stiffness (MRE)analysis was smaller than for other endpoints. Mean liver stiffness, asmeasured by MRE, decreased in all groups (see FIG. 3A). BMS-986036 10 mgQD and 20 mg QW groups compared with placebo had a greater percentage ofpatients with a ≥15% decrease in liver stiffness (see FIG. 3B). Thepercentages of subjects with ≥15% relative reduction were 36%, 33%, and7% for the BMS-986036 10 mg QD regimen, the BMS986036 20 mg QW regimen,and the placebo regimen, respectively. MRE images from a patient whoexperienced a reduction in liver stiffness following BMS-986036treatment are shown in FIG. 4 .

Adiponectin: Higher adiponectin levels are associated with improvementsin steatosis, inflammation, and fibrosis. Significant increases inadiponectin were observed following treatment with BMS-986036 10 mg QD(p=0.003) and 20 mg QW (p=0.003) compared with placebo (see FIG. 5 ),with mean levels peaking at Day 29 and then trending downward towardBaseline. At Day 112, the adiponectin mean percent change from Baselinewas 19% and 20% for the BMS-986036 20 mg QW and 10 mg QD treatmentgroups, respectively, compared with −1.1% for the placebo group,resulting in significant treatment differences (p=0.0071 for BMS-98603610 QD, 0.0072 for BMS-986036 20 QW).

Pro-C3: At baseline, mean serum PRO-C3 levels were comparable acrosstreatment groups, as shown in Table 3. Most patients (67% [28/42] hadbaseline Pro-C3 levels of <20 ng/mL as shown in FIGS. 6A-6B. Patientstreated with BMS-986036 10 mg QD and 20 mg QW had significantly reducedserum PRO-C3 levels compared with placebo (p<0.0001 and p=0-0093,respectively) at Day 112 from Baseline (see FIG. 7A). A maximal decreasewas obtained at Day 57 for BMS-986036 10 mg QD groups and sustained forthe rest of treatment period. There was no significant difference in thepercent changes between BMS-986036 20 mg QW and BMS-986036 10 mg QDgroups at Day 112. Furthermore, BMS-986036 10 mg QD and 20 mg QW groupscompared with placebo had a significantly greater percentage of patientswith a ≥15% relative reduction in serum PRO-C3 levels (QD: p=0.0025; QW:p=0.0063) (see FIG. 7B). 15% was chosen based on the precision of theassay. The percentages of subjects with ≥15% relative reduction were64%, 60%, and 18%, for the BMS-986036 10 mg QD regimen, the BMS-98603620 mg QW regimen, and the placebo regimen, respectively (Table 5). Nodifference in Pro-C3 response to treatment was observed by Baselinefibrosis stages.

TABLE 5 Change from Baseline at Week 16 - Pro-C3 Number (%) of SubjectsPercent Change from BMS 10 mg daily BMS 20 mg weekly Placebo dailyBaseline* at Week 16 N = 25 N = 24 N = 26 Pro-C3 >=15% IMPROVEMENT 14/22(63.6) 12/20 (60.0) 4/22 ( 18.2) 90% CI (0.467, 0.805) (0.419, 0.780)(0.064, 0.369) ODDS RATIO (90% CI) 7.88 (2.46, 25.26) 6.75 (2.08, 21.95)P-VALUE (ONE-SIDED) 0.0025 0.0063 *Baseline is defined as lastnon-missing result with a collection date-time less than the date-timeof the first active dose of study medication. Fisher exact test isconducted for this analysis.

ALT/AST: ALT and AST are biomarkers of liver injury. The mean absolutevalues of ALT and AST at Baseline and Week 16 are set forth in Table 6.

TABLE 6 Mean absolute values of ALT and AST at Baseline and Week 16BMS-986036 Placebo 10 mg QD 20 mg QW (n = 26) (n = 25) (n = 24) ALT,U/L, mean (SD) Baseline 80 (51) 66 (37) 70 (33) Week 16  73 (55)*  39(18)*  47 (30)^(†) AST, U/L, mean (SD) Baseline 58 (49) 48 (23) 52 (22)Week 16  47 (29)*  29 (11)*  35 (12)^(†) ALT = alanine aminotransferase.AST = aspartate aminotransferase. QD = once daily. QW = once weekly. SD= standard deviation. *n = 24. ^(†)n = 22.A robust decrease of ALT and AST was observed for both BMS-986036 dosegroups starting from Day 29 (see FIGS. 8A-8B). A maximal decrease at Day43 was observed for 10 mg QD group and at Day 57 for 20 mg QW group. Atrend of more decrease was observed in 10 mg QD group compared to 20 mgQW group (see FIGS. 8A-8B). In both treatment groups, aminotransferasedecreases were observed by Day 15 and a stable nadir was reached by Day57; this nadir continued through the end of treatment. In the placebogroup, ALT and AST did not change substantially from baseline.

Fasting lipids: NASH is frequently associated with dyslipidemia. BMS986036 at 10 mg QD and 20 mg QW resulted in improvements in TGs and HDL.HDL levels increased from Baseline for both 20 mg QW and 10 mg QDgroups, while there was no changes in HDL for placebo (see FIG. 9B). Ingeneral, fasting triglyceride levels have high variability with largestandard deviations. However, fasting triglyceride levels were decreasedin both BMS-986036 dose groups compared to baseline, with mean levelsdecreased at Day 29 to Day 86, levels then trended upwards towardBaseline at Day 112 (see FIG. 9C). In addition, a decrease in LDL wasobserved for 10 mg QD group, while no significant decrease in LDL wasobserved for 20 mg QW and placebo groups (see FIG. 9A). CalculatedIndices Related to NASH: Fibrosure fibrosis scores were reduced fromBaseline in the BMS-986036 groups, while no reduction was observed inthe placebo group.

BMS-986036 was generally well tolerated. There were no deaths ordiscontinuations due to adverse events, as set forth in Table 7.

TABLE 7 Safety Summary BMS-986036 Placebo 10 mg QD 20 mg QW Event, n (%)(n = 26) (n = 25) (n = 24) Deaths 0 0 0 Discontinuation due to AEs 0 0 0Serious AEs 1 (4) 1 (4) 0 Treatment-related SAEs 0 0 0 Overall AEs 15(58) 18 (72) 13 (54) Most frequent AEs Diarrhoea 2 (8)  3 (12)  5 (21)Nausea 2 (8)  4 (16)  3 (13) Fatigue  5 (19) 1 (4) 0 Headache  3 (12) 1(4) 2 (8) Urinary tract infection 2 (8) 1 (4)  3 (13) Frequent bowel 0 5 (20) 0 movements Cough 2 (8) 1 (4) 1 (4) Injection site bruising 0 2(8) 2 (8) Grade 3 laboratory 2 (8) 1 (4) 1 (4) abnormalities ofincreased ALT* AE = adverse event. ALT = alanine aminotransferase. SAE =serious adverse event. *There were no grade 4 laboratory abnormalitiesobserved.The most frequently reported AEs were diarrhoea (13%, [8/49 patientstreated with either dose of BMS-986036 and 2/26 patients who receivedplacebo]) and nausea (12%, [7/49 patients treated with either dose ofBMS-986036 and 2/26 patients who received placebo]). There were 2serious adverse events: one patient in the BMS-986036 10 mg QD groupexperienced worsened depression and attempted suicide, and one patientwho was randomized to the placebo group received the wrong study drugduring the placebo lead-in period. There were 28 adverse events amongthe following composite gastrointestinal AEs: diarrhoea, nausea,frequent bowel movements, upper abdominal pain, and vomiting. Of these28 events, 11 were considered treatment-related. The frequency ofgastrointestinal adverse events was higher in patients treated withBMS-986036 compared with patients who received placebo. However, therewas no clear association between AE frequency and BMS-986036 dose. Allgastrointestinal events were considered mild or moderate. Of thetreatment-related gastrointestinal AEs, only 1/11 required treatment forresolution (1 patient who experienced frequent bowel movements wastreated with Bifidobacterium infantis). Injection site bruising,erythema, or reactions were reported by 5%, 4%, and 3% of all patients,respectively. These events were mostly mild, transient, and did notrequire treatment. In total, 5 of 75 patients (7%) experienced atreatment-emergent grade 3 laboratory abnormality. There was 1 event ofgrade 3 high glucose (increase from grade 2 at baseline in a patientwith T2DM treated with BMS-986036 20 mg QW) and 4 events of grade 3 ALTelevations (1 increase from grade 1 at baseline in a patient treatedwith BMS-986036 20 mg QW, 3 increases from grade 2 at baseline in 2patients who received placebo and 1 patient who received BMS-986036 10mg QD), which improved despite continued dosing. No grade 4 laboratoryabnormalities were observed. No clinically relevant changes in ECGintervals or vital signs following treatment with BMS-986036 wereobserved. Mean BMDs of the femur (FIG. 10A), hip (FIG. 10B), and spine(FIG. 10C), as measured by DXA, were not meaningfully different ineither BMS-986036 dose group compared with placebo at either Week 16 or6 months after the end of treatment.

At time points up to study discharge on Day 142, anti-BMS-986036 andanti-FGF21 antibodies were detected in 62.5% (15/24) of patients treatedwith BMS-986036 20 mg QW and in 92% (23/25) of patients treated withBMS-986036 10 mg QD. Antibody titres were generally low (the majoritywere <64), and they were not associated with immune-related adverseevents, injection site reactions, or changes in pharmacokinetic orpharmacodynamic profiles. At the time of post-study follow up visitsthat occurred 6 months after the end of treatment, <50% of patients hadpositive titres for anti-BMS-986036 and/or anti-FGF21 antibodies, andonly one patient had an antibody titre ≥64.

C. Discussion

Compared with placebo, patients with NASH who were treated with 10 mg QDor 20 mg QW BMS-986036 had significantly decreased absolute hepatic fatfraction and significantly increased levels of adiponectin. Mean lipidvalues and markers of liver injury (ALT and AST) were decreased in the10 mg QD and 20 mg QW groups compared with baseline values. Mean liverstiffness decreased in all treatment groups. In both the 10 mg QD and 20mg QW groups, patients had significantly decreased levels of PRO-C3 (abiomarker of fibrosis) compared with placebo. Overall, the effects ofdaily and weekly dosing appear to be similar and treatment with eitherBMS-986036 dose was generally well tolerated.

In both BMS-986036 treatment groups, patients achieved a significantabsolute reduction in hepatic fat fraction as measured by MRI-PDFF andover half of the patients achieved ≥30% relative reduction. In aprevious study, histologic response (≥2-point reduction in NAS) wasassociated with a mean relative reduction in hepatic fat fraction of 29%as measured by MRI-PDFF (see Patel J, et al. Therap. Adv. Gastroenterol.2016; 9(5): 692-701). These data, along with future histology-basedanalyses, further support the clinical utility of MRI-PDFF as anon-invasive method of evaluating steatosis in NASH patients.

Treatment with either dose of BMS-986036 was associated with significantimprovements in adiponectin consistent with what has been previouslyreported in trials of other FGF21 analogues for patients with T2DM (seeGaich G, et al., Cell. Metab. 2013; 18(3): 333-40 and Talukdar S, etal., Cell Metab 2016; 23(3): 427-40). In patients with NASH, adiponectinlevels can be at least 50% lower than in healthy individuals (see PaganoC, et al., Eur. J. Endocrinol. 2005; 152(1): 113-8), suggesting thatadiponectin has important hepatic effects that are protective againstNASH. Adiponectin knockout mice placed on a high fat diet have increasedintrahepatic triglycerides, hepatocyte ballooning and fibrosis andadiponectin administration reduces steatosis, reduce inflammation, andhas beneficial effects on lipid metabolism (see Asano T, et al., JGastroenterol Hepatol 2009; 24(10): 1669-76 and Xu A, et al., J. Clin.Invest. 2003; 112(1): 91-100). Furthermore, adiponectin antagonizeshepatic stellate cell activation, a process critical to fibrogenesis,and it has anti-fibrotic effects in TGF-beta-stimulated fibroblasts andin mice with CCl₄-induced liver fibrosis (see Shafiei M S et al., Am JPathol 2011; 178(6): 2690-9; Fang F, et al., Arthritis Res Ther 2012;14(5): R229; and Kumar P, et al., PLoS One 2014; 9(10): e110405).

Approximately ⅓ of patients in the 10 mg QD and 20 mg QW groups achieved≥15% relative reduction in liver stiffness as measured by MRE, anon-invasive imaging technique. A 15% relative reduction in liverstiffness measured by MRE has been associated with significant reductionin serum markers of fibrosis (see Loomba R, et al., J Hepatol 2017;66(Suppl 1): S671). While the small sample size limits theinterpretability of these results, it is encouraging that this thresholdcan be reached with a relatively short treatment duration. MRE is apromising imaging biomarker of fibrosis. It is reproducible and has ahigh rate of technical success.

At Week 16, BMS-986036-treated patients also had significantly decreasedlevels of PRO-C3, a measure of type III collagen formation, comparedwith placebo. In patients with NASH, the levels of PRO-C3 have beenassociated with NASH disease activity and fibrosis stage (see Leeming DJ, et al., J Hepatol 2017; 66(Suppl 1): S154 and Leeming D J, et al.,Plasma collagen III type III (PRO-C3) levels associate with severity ofhistological features of non-alcoholic steatohepatitis and fibrosiswithin the screening population from the CENTAUR study. NASH BiomarkersWorkshop 2017 2017; Presented on Saturday, May 5, 2017). In patientswith chronic hepatitis C virus infection, PRO-C3 has been shown tocorrelate with the severity of liver fibrosis, and high baseline PRO-C3levels have been associated with increased fibrotic disease progression(see Nielsen M J, PLoS One 2015; 10(9): e0137302). The serum PRO-C3assay provides an accurate, non-invasive method to identifyanti-fibrotic treatment response (see Karsdal M A, et al., Am J PhysiolGastrointest Liver Physiol 2016; 311(6): G1009-17). Furthermore,longitudinal decreases in PRO-C3 have been correlated with improvementsin hepatic fibrosis, as assessed by biopsy (see Luo Y., et al., JHepatol 2017; 66(Suppl 1): S676). Given the above, the reduction inPRO-C3 observed in BMS-986036-treated patients seems to be indicative ofan anti-fibrotic treatment effect.

In this study, no substantial changes in body weight were observed inpatients with NASH after 16-week treatment with BMS-986036. AlthoughFGF21-induced weight loss has been shown in animal studies, the absenceof weight loss in humans is consistent with observations from a previoustrial of BMS-986036 in obese patients with T2DM (see Kharitonenkov A, etal., Endocrinology 2007; 148(2): 774-81; Coskun T, et al., Endocrinology2008; 149(12): 6018-27; Xu J, et al., Am J Physiol Endocrinol Metab2009; 297(5): E1105-14; Charles E, et al., Hepatology 2016; 64(Suppl 1):17A). The reason for the differences in weight loss in animal studiesversus clinical trials is unclear. However, there may be a distinctdifference in the FGF21 physiology of humans versus rodents andnon-human primates.

Overall, daily or weekly doses of BMS-986036 were generally safe andwell tolerated, with no deaths, treatment-related serious adverseevents, or discontinuations due to injection burden or AEs. The mostfrequently reported adverse events were gastrointestinal in nature(diarrhoea and nausea), which is consistent with previous observationsin other studies of FGF21 analogues (see Talukdar S, et al., Cell Metab2016; 23(3): 427-40 and Fang F, et al., Arthritis Res Ther 2012; 14(5):R229). These adverse events were generally mild and did not requiretreatment. Similarly, injection site reactions were mostly mild,transient, and did not require treatment. In contrast to a recent studyof an intravenously administered, long-acting FGF21 analog, no obviouschanges in heart rate or blood pressure were observed with BMS-986036treatment; this is consistent with results from the BMS-986036first-in-human study in which these parameters were intensivelymonitored (see Kim A M, Somayaji V R, Dong J Q, et al., Diabetes ObesMetab 2017; 19(12): 1762-72 and Charles E, et al., Hepatology 2016;64(Suppl 1): 546A). In animal models, FGF21 administration has beenassociated with changes in BMD (see Wei W, et al., Proc Natl Acad SciUSA 2012; 109(8): 3143-8). However, in this study, mean BMD (determinedby DXA) did not meaningfully change for either BMS-986036 treatmentgroup, compared with placebo, from baseline through the end oftreatment, or through 6 months of follow-up.

Given that BMS-986036 is a pegylated human FGF21 mimetic, the potentialexists that it may elicit immunogenic responses in some patients.Immunogenicity studies showed that anti-BMS-986036 and anti-FGF21antibodies were detectable in over half of the patients treated withBMS-986036 20 mg QW and in over 90% of patients treated with BMS-98603610 mg QD. These antibody titres were generally low, were not associatedwith immune-related adverse events or injection site reactions, and weredeclining in many patients by the time of follow-up visits. There was noevidence to suggest breakage in immune tolerance to endogenous FGF21since titres to endogenous FGF21 antibodies decreased afterdiscontinuation of treatment. Together, these data suggest thatBMS-986036 does not elicit clinically meaningful immunogenicity whenadministered QD or QW over a 16-week period.

The major findings of this study further extend knowledge gained fromwith an earlier 12-week, phase 2 trial of BMS-986036 in obese T2DMpatients (MB130-002) (see Charles E, Neuschwander-Tetri B, et al.,Hepatology 2016; 64(Suppl 1): 17A). Obesity and T2DM are two major riskfactors for the development of NAFLD, including NASH. In MB130-002, mostpatients were likely to have underlying NAFLD. 97% of patients had aFatty Liver Index (FLI) ≥60, a cutoff indicative of the presence offatty liver (see Bedogni G, et al., BMC Gastroenterol 2006; 6: 33). Inthese patients, BMS-986036 also improved lipids, adiponectin and PRO-C3levels. Importantly, the safety profile of BMS-986036 was comparable inMB130-002 and the instant study, and it was characterized by anincreased frequency of predominantly mild gastrointestinal symptoms, noinstances of drug-induced liver injury, no clinically meaningful changesin ECG or vital signs, and no apparent effect on bone density (asmeasured by DXA) over a 16-week time period. Taken together, datasuggest that BMS-986036 treatment is well tolerated and has positiveeffects on liver and metabolic parameters in patients with NASH, as wellas in patients with risk factors for NASH. In summary, BMS-986036 wasgenerally safe and well tolerated when administered by SC injection atdoses of BMS-986036 20 mg QW or BMS-986036 10 mg QD for 16 weeks in NASHsubjects. Results suggest that BMS-986036 has beneficial effects onsteatosis, liver injury, and fibrosis in NASH: Both BMS-986036 10 mg QDand BMS-986036 20 mg QW regimens, compared with placebo, significantlyreduced hepatic fat fraction at Week 16. Relative to placebo, BMS-986036QD and QW were associated with improvements in biomarkers of fibrosis(MRE and Pro-C3), metabolic parameters (adiponectin and lipids), andmarkers of hepatic injury (ALT and AST). Improvements in MRI-PDFF,Pro-C3, LDL, ALT, and AST exhibited dose-dependence. For each marker(except for LDL), the majority of the maximally observed treatmenteffect difference was achieved with BMS-986036 20 mg QW. Ctrough ofBMS-986036 (C-terminal intact and Total) was higher for 10 mg QDrelative to 20 mg QW. Ctrough levels remained stable after the steadystate achieved.

Example 2: Baseline Serum Pro-C3 Predicts Response to BMS-986036: ASecondary Analysis of a Multi-Center Clinical Trial in NASH

The objective of this post hoc analysis of the clinical trial fromExample 1 was to assess Baseline predictors of treatment response toBMS-986036.

Post hoc analyses assessed the relationship between Baselinecharacteristics and changes in markers of metabolism, steatosis, liverinjury, and fibrosis. Evaluations included type 2 diabetes mellitus(T2DM) status, liver biopsy grade (NAFLD Activity Score [NAS]) andfibrosis stage (NASH CRN criteria) and serum Pro-C3 levels (≥20 vs <20ng/mL). Hepatic and metabolic markers were evaluated at Baseline andWeek 16 and included liver fat (MRI-PDFF), liver stiffness (MRE) andserum biomarkers: Pro-C3, ALT, AST, adiponectin, LDL, HDL, hyaluronicacid (HA), PAI-1, and CK-18 (cytokeratin-18). For the purposes of thisanalysis, BMS-986036 groups (20 mg QW [n=23] vs 10 mg QD [n=25]) werecombined, and placebo group (n=26) was analyzed separately. Statisticalcomparisons were made using Kruskal-Wallis tests.

Mean baseline Pro-C3 levels were generally consistent across BMS-986036treatment groups, in which most patients (67%, [28/42]) had baselinePro-C3 levels of <20 ng/mL. In BMS-986036-treated patients (n=48),Baseline serum Pro-C3 level ≥20 ng/mL, compared with <20 ng/mL, wasassociated with greater reduction in MRE (median % change: −16.4% vs6.0%, P=0.0198) and greater decreases in Pro-C3 levels (median % change:−34.9% vs −23.1%, P=0.08) (FIG. 11A-11B). Baseline Pro-C3 levels did notpredict BMS-986036-treatment response assessed via other hepatic ormetabolic biomarkers. Other Baseline characteristics, including T2DMstatus, NAS, or fibrosis stage did not clearly impact biomarkerresponses in BMS-986036-treated patients. In the placebo group, noBaseline characteristics, including Pro-C3 levels, were clearlyassociated with differences in the biomarker responses evaluated.

In this Phase 2 clinical trial in NASH, high Baseline Pro-C3 levels wereassociated with improvements in biomarkers of fibrosis and liverstiffness (Pro-C3 and MRE) in BMS-986036-treated patients. Conversely,other Baseline characteristics, including fibrosis severity, did notappear to predict response to BMS-986036 treatment. These resultssupport the use of serum Pro-C3 as a predictor for treatment response toBMS-986036 and as a potential surrogate marker to identify the idealNASH patients for treatment.

On the other hand, patients with a reduction in hepatic fat fraction≥30% versus patients with <30% had significantly greater decreases inPro-C3, ALT, AST, and CK-18 (FIGS. 12A-12D). In addition, a reduction inliver stiffness of ≥15% compared with reduction <15% was associated withdecrease in Pro-C3, HA and increase in HDL (FIGS. 13A-13C).

SEQUENCE SUMMARY SEQ ID NO: SEQUENCE SEQ ID NO: 1HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAH (correspondingLEIREDGTVGGAADQSPESLLQLKALKPGVIQ to SEQ ID NO:ILGVKTSRFLCQRPDGALYGSLHFDPEACSFR 1 of ELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPU.S. Pat. No. APRGPARFLPLPGLPPAPPEPPGILAPQPPDV 9,079,971)GSSDPLSMVGPSQGRSPSYAS SEQ ID NO: 2 MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEA(corresponding HLEIREDGTVGGAADQSPESLLQLKALKPGVI to SEQ ID NO:QILGVKTSRFLCQRPDGALYGSLHFDPEACSF 201 of RELLLEDGYNVY(pAF)SEAHGLPLHLPGNKSU.S. Pat. No. PHRDPAPRGPARFLPL PGLPPAPPEPPGILA 9,434,778)PQPPDVGSSDPLSMVGPSQGRSPSYAS

1. A method for treating a patient having Nonalcoholic Steatohepatitis(NASH) comprising: (a) determining the serum Pro-C3 level in a bloodsample from the patient and (b) administering to the patient a modifiedFibroblast growth factor 21 (FGF-21) in an amount and with a frequencysufficient to treat NASH, if the serum Pro-C3 level is greater than 10ng/ML. 2.-4. (canceled)
 5. A method for monitoring responsiveness of apatient having NASH to treatment with a modified FGF-21, the methodcomprising: determining the serum Pro-C3 level in a blood sample fromthe patient obtained during or after treatment, wherein: a decreasedserum Pro-C3 level in the blood sample from the patient obtained duringor after treatment, as compared to the serum Pro-C3 level in a bloodsample from the patient obtained prior to treatment with the modifiedFGF-21, indicates that the patient is responsive to treatment with themodified FGF-21.
 6. The method of claim 1, wherein the serum Pro-C3level prior to administration to the patient of a modified Fibroblastgrowth factor 21 (FGF-21) is greater than about 11 ng/ML, 12 ng/ML, 13ng/ML, 14 ng/ML, 15 ng/ML, 16 ng/ML, 17 ng/ML, 18 ng/ML, 19 ng/ML, 20ng/ML, 21 ng/ML, 22 ng/ML, 23 ng/ML, 24 ng/ML, or 25 ng/ML.
 7. Themethod of claim 6, wherein the serum Pro-C3 level is greater than about15 ng/ML.
 8. The method of claim 1, wherein the Pro-C3 level or levelsare measured by an FDA-approved test.
 9. The method of claim 1, whereinthe Pro-C3 level or levels are measured by using an immunoassay,immunochemistry, immunohistochemistry assay, nucleoprobe assay, in situhybridization, fluorescent RNA probes, RT-PCR, microarray transcriptionassay, or RNA transcription assay.
 10. The method of claim 9, whereinthe immunoassay is an enzyme-linked immunosorbent assay (ELISA) or aradioimmunoassay (RIA).
 11. The method of claim 1, wherein the modifiedFGF-21 comprises the polypeptide of SEQ ID NO:1, except that an aminoacid in the polypeptide is substituted by a non-naturally encoded aminoacid, wherein: (a) said non-naturally encoded amino acid is at aposition corresponding to residue 108 of SEQ ID NO:1; and (b) saidnon-naturally encoded amino acid comprises para-acetyl phenylalaninelinked to a polymer comprising a poly(ethylene glycol).
 12. The methodof claim 1, wherein the modified FGF-21 comprises the polypeptide of SEQID NO:1 except that the amino acid at position 108 of SEQ ID NO:1 issubstituted by a non-naturally encoded amino acid, wherein: (a) saidnon-naturally encoded amino acid comprises para-acetyl phenylalanine,and (b) said non-naturally encoded amino acid is linked to a polymercomprising a poly(ethylene glycol) having an average molecular weight ofabout 30 kDa.
 13. The method of claim 11, wherein said poly(ethyleneglycol) has an average molecular weight of about 30 kDa.
 14. The methodof claim 11, wherein said non-naturally encoded amino acid is linked tosaid polymer through an oxime linkage.
 15. The method of claim 1,wherein the modified FGF-21 comprises SEQ ID NO:2.
 16. The method ofclaim 15, wherein the modified FGF-21 comprises a poly(ethylene glycol)having an average molecular weight of about 30 kDa.
 17. The method ofclaim 16, wherein the poly(ethylene glycol) is linked to para-acetylphenylalanine.
 18. The method of claim 1, wherein the modified FGF-21 isadministered at a once weekly dose of 20 mg or a once daily dose of 10mg.
 19. The method of claim 1, comprising administration of a secondtherapeutic agent.
 20. The method of claim 1, wherein the treatment: (a)results in a decrease in serum Pro-C3 levels in the patient; (b)produces a shift toward normal serum levels of Pro-C3 in the patient;(c) results in a reduction in liver stiffness in the patient compared tothe patient's liver stiffness prior to treatment, wherein liverstiffness is assessed by magnetic resonance elastography (MRE); and/or(d) results in a reduction in hepatic fat fraction in the patientcompared to the patient's hepatic fat fraction prior to treatment,wherein hepatic fat fraction is as assessed by magnetic resonanceimaging-estimated proton density fat fraction (MRI-PDFF). 21.-26.(canceled)
 27. The method of claim 1, wherein the treatment produces atleast one therapeutic effect in the patient selected from the groupconsisting of a reduction or cessation in fatigue, malaise, weight loss,and/or right upper quadrant abdominal discomfort. 28.-30. (canceled)